Anti cancer mechanisms of indirubin 3 monoxime

... 1 .3. 4 Other regulatory mechanisms 30 1 .3. 5 Apoptosis and cancer 30 1 .3. 6 TRAIL signaling pathway as potential therapeutic target 32 Objectives of the study Chapter Materials and Methods 33 34 ... accompanying p 53 up-regulation 51 3. 4 I3M causes Bid cleavage 55 3. 5 I3M induces Bax conformational changes 59 3. 6 I3M induces cytochrome c release 63 v 3. 7 3. 8 Overexpression of Bcl-2 or CrmA... in response to I3M treatment 46 Figure 3. 2 Growth inhibitory effects of I3M on cancer cells 47 Figure 3. 3 Time- and dose-dependent apoptosis in cancer cell lines 48 Figure 3. 4 I3M-induced caspase

ANTI-CANCER MECHANISMS OF INDIRUBIN-3’-MONOXIME SHI JIE (B. Sc. with Honors, Life Sciences, National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF COMMUNITY, OCCUPATIONAL AND FAMILY MEDICINE, YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to express my deepest respect and acknowledgements to my supervisor, Associate Prof. Shen Han-Ming, for his consistent and invaluable guidance throughout my M.Sc. study. He is the person who always encourages me, provides professional comments and guides me to the right way of doing scientific research. What I have learned from him will benefit my career and life. I would also like to extend my sincere gratitude and appreciation to: Prof Ong Choon Nam for his kind comments and support Prof. David Koh, Head of the department, for his general kind support during the course of this study. Mr. Ong Her Yam, Mr. Ong Yeong Bing, Ms Su Jin and Ms Zhao Min for their kind help in the process of laboratory work. Dr. Shi Ranxin, Dr. Huang Qing, Dr. Zhang Siyuan, Dr. Luo Guodong, and Ms Zhou Jing for their critical discussions, invaluable comments and consistent help during whole course of my study. Dr. Lai Jiaping and Dr. Ong Eng Shi, for their critical comments on the project. All other staff in Department, for their general and unselfish help. National University of Singapore, for the research scholarship Especially, I would like express my deepest appreciation to my family members and friends for their love, understanding and support. ii TABLE OF CONTENTS Title page i Acknowledgements ii Table of contents iii Summary vii List of Publications viii List of Figures ix List of Tables xi List of Abbreviations xii 1 Chapter 1 1.1 1.2 Introduction Indirubin and its derivatives 1 2 1.1.1 General introduction 2 1.1.2 Chemical structures and purification 4 1.1.3 Cellular absorption and metabolism 5 Pharmacological mechanisms of Indirubin and it derivatives 5 1.2.1 Anti-inflammatory activity 5 1.2.2 Anti-cancer activity 6 1.2.2.1 Clinical trials 7 1.2.2.2 In vitro experimental evidence 7 1.2.2.2.1 Cell cycle 8 1.2.2.2.2 Cytotoxicity 11 1.2.2.2.3 Sensitization 13 1.2.2.2.4 Anti-angiogenesis 14 iii 1.2.2.3 In vivo Animal Model 1.2.3 Molecular targets of I3M 1.3 1.4 16 16 1.2.3.1 Cyclin-dependent kinase (CDK) 16 1.2.3.2 Src kinase and Stat3 signalling pathway 18 1.2.3.3 Aryl hydrocarbon receptor (AhR) 18 1.2.3.4 Glycogen synthetase kinase-3 (GSK3) 19 1.2.3.5 c-Jun NH2-terminal kinase (JNK) 20 1.2.3.6 Fibroblast growth factor receptor 1 (FGFR1) 20 Apoptosis 21 1.3.1 General introduction 21 1.3.2 Apoptosis pathways 22 1.3.2.1 Receptor-mediated apoptosis 25 1.3.2.2 Mitochondrial-mediated apoptosis 27 1.3.2.3 Type I and type II cells 28 1.3.3 Bcl-2 family 28 1.3.4 Other regulatory mechanisms 30 1.3.5 Apoptosis and cancer 30 1.3.6 TRAIL signaling pathway as potential therapeutic target 32 Objectives of the study Chapter 2 Materials and Methods 33 34 2.1 Reagents, chemicals and plasmids 35 2.2 Cell culture 35 2.3 Growth inhibition test: MTT 36 2.4 Western blot 36 iv 2.5 Detection of apoptosis 37 2.5.1 DAPI staining 37 2.5.2 PI staining followed by flow cytometry 37 2.6 Measurement of caspase activity 38 2.7 Measurement of surface expressions of death receptors 38 2.8 Transfection 39 2.8.1 Transient transfection of siRNA 39 2.8.2 Stable transfection of vectors expressing Bid siRNA 39 2.8.3 Detection of Bcl-2 or CrmA transfected cells using flow cytometry 40 2.9 Detection of Bax conformational change 40 2.9.1 Immunofluorescence 40 2.9.2 Immunoprecipitation 41 2.10 Subcellular fractionation 42 2.11 Long-term clonogenic assay 42 2.12 Statistical analysis 42 Chapter 3 3.1 Results 44 I3M induces apoptosis in a time- and dose-dependent manner in human cancer cells 45 3.2 I3M leads to caspase activation 45 3.3 I3M induces increased surface expression of death receptors accompanying p53 up-regulation 51 3.4 I3M causes Bid cleavage 55 3.5 I3M induces Bax conformational changes 59 3.6 I3M induces cytochrome c release 63 v 3.7 3.8 Overexpression of Bcl-2 or CrmA partially blocks I3M-induced apoptosis 63 I3M sensitizes cancer cells to TRAIL–induced apoptosis 68 Chapter 4 Discussions and conclusions 4.1 I3M induce apoptosis in HeLa, HepG2 and HCT116 4.2 Apoptosis induced by I3M recruits extrinsic pathways with type II 72 73 response 74 4.3 Critical role of pro-apoptotic Bcl-2 family members 77 4.4 Sensitization of TRAIL-induced apoptosis 78 4.5 Conclusions 79 4.6 Directions for future study 79 Chapter 5 References 81 vi SUMMARY Indirubin-3’-monoxime (I3M) is a derivative of indirubin, an active component from a Chinese medicinal recipe for the treatment of leukemia. Indirubin and its derivatives, a group of bis-indole alkaloid, have exhibited strong growth inhibitory effects on various human cancer cells. In the attempt to reveal the mechanism of action of indirubins, various biological activities of indirubin and its derivatives have been discovered. To investigate the anti-cancer effect of I3M, a study was done to evaluate the apoptotic machinery involved by focusing on the role of the pro-apoptotic Bcl-2 family members in I3M-induced apoptotic cell death. First, the cytotoxicity of I3M in three different types of human cancer cells was confirmed — cervical cancer-HeLa, hepatoma-HepG2 and colon cancer-HCT116 through flow cytometry analysis (after propidium iodide staining) and MTT assays. I3M -induced apoptosis was detected in a time- and dose-dependent manner in the above mentioned human cancer cells, based on 4',6-diamidino-2-phenylindole (DAPI) staining, flow cytometry analysis and PARP cleavage. Caspase-8, caspase-9 and caspase-3 cleavage were observed in I3M-induced apoptosis in HeLa. Caspase 3/7 activity in the three cell lines was also measured. The participation of various caspases in I3M-induced apoptosis was examined by using respective synthetic inhibitors. An increased surface expression level of death receptor 4 and 5 (DR4 and DR5) was detected in HeLa cells. A time-dependent increase in the total protein level of DR4 and DR5 was also observed. Interestingly, concurrent up-regulation of p53 protein level and its transcriptional activity as indicated by increased p21 correlated vii with the up-regulation of DR4 and DR5, suggesting an important role of p53 in the initiation of the death receptor pathway. One important observation was that caspase-8 activation resulted in Bid cleavage, followed by Bax conformational change, and cytochrome c release. Stable knockdown of Bid partially protected I3M-induced apoptosis by blocking Bax conformational change. Also, transient overexpression of a viral caspase inhibitor (CrmA) or Bcl-2 partially protected I3M-induced apoptosis. Furthermore, I3M was shown to sensitize TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cell lines according to flow cytometry analysis, DAPI staining, and long-term colony formation assay. In conclusion, I3M-induced apoptosis was found to engage both extrinsic and intrinsic pathways involving the pro-apoptotic Bcl-2 family members (Bid and Bax) which play a critical role. In addition, there was evidence showing the sensitizing effect of I3M on TRAIL-induced apoptosis, which will form the basis of future studies. LIST OF PUBLICATIONS The following paper has been published: Shi J and Shen HM. Indirubin-3’-monoxim induced apoptosis in human tumor cells Bcl-2 family members. Biochem Pharmacol 2008 May 1;75(9):1729-42. Epub 2008 Mar 10 viii LIST OF FIGURES Figure 1.1 Qing Dai and chemical structures of indirubin derivatives. 3 Figure 1.2 Cell cycle and major regulator proteins 9 Figure 1.3 A schematic representation of the effect of I3M on TNF-induced NFκB activation and apoptosis 15 Figure 1.4 Evolutionarily conserved cell death pathways 23 Figure 1.5 Apoptotic pathways in mammalian system 24 Figure 1.6 Extrinsic death receptor pathways 26 Figure 3.1 Morphological changes and nuclear condensation of cancer cells in response to I3M treatment 46 Figure 3.2 Growth inhibitory effects of I3M on cancer cells 47 Figure 3.3 Time- and dose-dependent apoptosis in cancer cell lines 48 Figure 3.4 I3M-induced caspase activation. 50 Figure 3.5 Inhibition of I3M-induced apoptosis by synthetic caspase inhibitors 52 Figure 3.6 Inhibition of I3M-induced caspase activation by synthetic caspase inhibitors 53 Figure 3.7 I3M-induced enhanced surface expressions of DR4 and DR5 54 Figure 3.8 Bid cleavage in response to caspase activation in I3M-induced apoptosis. Figure 3.9 Protection conveyed by Bid siRNA transient transfection against I3Minduced apoptosis Figure 3.10 57 Protection conveyed by Bid knockdown against I3M-induced apoptosis Figure 3.11 56 58 Bax conformational change following caspase-8 activation in I3M- ix treated HeLa cells. 60 Figure 3.12 I3M-induced cytochrome c release 64 Figure 3.13 Ectopic expression of Bcl-2 or CrmA protects against I3M-induced Figure 3.14 apoptosis 65 I3M sensitizes human cancer cells to TRAIL-induced apoptosis 69 x LIST OF TABLES Table 1.1 Table 1.2 Growth inhibitory effects of indirubin and its derivatives on various human cancer cell lines. 12 Kinase targets of indirubin and its derivatives 17 xi LIST OF ABBREVIATIONS ac-IETD-CHO, N-acetyl-Ile-Glu-Thr-Asp-CHO (aldehyde) ac-LEHD-CHO, N-acetyl-Leu-Glu-His-Asp-CHO (aldehyde) AhR Aryl hydrocarbon receptor AIF apoptosis inducing factor Apaf-1 apoptotic protease-activating factor 1 CARD caspase recruitment domains CDK cyclin-dependent kinases CHX cycloheximide c-IAP cellular inhibitor of apoptosis protein CML chronic myelocytic leukaemia CrmA cytokine response modifier A DAPI 4',6-diamidino-2-phenylindole DD death domain DED death effector domain DISC death-inducing signaling complex DMSO dimethyl sulfoxide DR death receptor EDTA ethylenediaminetetraacetic acid FADD Fas-associated death domain FBS fetal bovine serum FGF-1 fibroblast growth factor 1 FGFR1 fibroblast growth factor receptor 1 xii FLIP FLICE (FADD-like IL-1β-converting enzyme) inhibitory protein GFP green fluorescence protein IκB inhibitor of κB IKK IκB kinase JNK c-Jun N-terminal kinase MAPK mitogen-activated protein kinases MPTP 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium NF-κB nuclear transcription factor-kappaB PARP poly(ADP-ribose) polymerase PI propidium iodide PI3K phosphatidylinositol 3'-kinase PMA paramethoxyamphetamine PMSF phenylmethylsulfonyl fluoride pRB retinoblastoma protein tBid truncated Bid TNFα tumor necrosis factor-α TNFR1 TNF receptor 1 TRADD TNF receptor-associated death domain TRAF2 TNF receptor-associated factor 2 TRAIL TNF-related apoptosis-inducing ligand XIAP X-linked inhibitor of apoptosis z-DEVD-FMK, z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-Fluoromethylketone z-VAD-fmk N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone xiii CHAPTER ONE INTRODUCTION 1 Chapter 1 Introduction 1.1 Indirubin and its derivatives 1.1.1 General introduction Scientific interest on indirubin originated from its role as an anti-leukemia agent, the active component of a traditional Chinese medicine preparation Danggui Longhui Wan, which has been used in China for the treatment of various chronic diseases for centuries (Eisenbrand et al., 2004; Xiao et al., 2002). Out of the 11 herbal constituents, Qing Dai (Indigo naturalis) was initially characterized as the active ingredient, a dark blue mixture isolated from the leaves of Baphicacanthus cusia (Acanthaceae), Polygonum tinctorium (Polygonaceae), Isatis indigotica (Brassicaceae), Indigofera suffrutticosa (Fabaceae) and Indigofera tinctoria (Fabaceae)(Hoessel et al., 1999) (Fig. 1.1A). Although a high level of indigo (blue dye) is present in the mixture, the antileukemic activity was attributed to the presence of red-coloured 3,2’-isomer indirubin (Wu and Fang, 1980; Zhang et al., 1979a; Zhang et al., 1979b; Zhang, 1983). Indirubin and its derivatives have exhibited strong growth inhibitory effects on various human cancer cells, manifested by either cell cycle arrest (G2/M) (Hoessel et al., 1999; Yamaguchi et al., 2002) or cytotoxicity (mainly apoptosis) (Nam et al., 2005; Perabo et al., 2006; Ribas et al., 2006; Yamaguchi et al., 2002). An in vivo study carried out in rat tumor model provides further evidence for indirubins’ anti-tumor activity (Kim et al., 2007). In the attempt to reveal the mechanism of action of indirubins, various biological activities of indirubin and its derivatives have been discovered. It has been well established that indirubin and indirubin-3’-monoxime (I3M) are strong inhibitors for cyclin-dependent kinases (CDKs) (Hoessel et al., 1999; Perabo et al., 2006). In addition, there is evidence suggesting that indirubin and I3M inhibit glycogen 2 A. Qing Dai Indigo Blue B. Figure 1.1 Qing Dai and chemical structures of indirubin derivatives. A. Photos of Indigo naturalis and indigo blue. B. Chemical structures of indigo, indirubin and indirubin derivatives (modified from Hoessel et al. 1999 complimentary data). 3 synthase kinase-3ß (GSK-3ß) (Leclerc et al., 2001; Meijer et al., 2003), and c-Src kinase (Stat3 signalling) (Nam et al., 2005), but activate aryl hydrocarbon receptor (AhR), a co-transcriptional factor (Adachi et al., 2001). It was reported that indirubin could suppress the nuclear factor kappa B (NF-κB) activation and hence sensitize tumor necrosis factor (TNF)-induced apoptosis (Sethi et al., 2006). More recently, I3M has been found to inhibit autophosphorylation of FGFR1 and stimulate ERK1/2 activity through long-term p38 MAPK activation (Zhen et al., 2007). 1.1.2 Chemical structure and purification Indirubin and its derivatives are a group of bis-indole alkaloid with their chemical structures shown in Fig. 1.1B. The presence of indirubin in the mixture prepared from Qing Dai is minute (0.11%)(Xiao et al., 2002); therefore synthesis of indirubin is required. Indirubin (also known as isoindigotin or indigo red) can be derived by oxidation and spontaneous dimerization of indoxyl and isatin, which are in turn derived from precursor conjugates in naturally occuring isatan B (from Isatis tinctoria) or indican (from Indigofera and Polygonum); the detailed process has been described by Meijer et al. in 1999. Due to the poor solubility of indirubin, indirubin-3’monoxime (I3M), a derivative of indirubin, is purchased from Sigma and studied in the project. Besides extraction from indigo dye-producing plants with more than 200 species, indirubin is also present in another dye ‘Tyrean purple’, which can be produced from more than 15 species of Muricidae mollusks (Meijer et al., 2003). In addition, certain wild-type and recombinant bacteria can also synthesize indirubin (with reference to Wu et al. 2005). Under pathological conditions including leukemia, indirubin can be detected in human urine, which can be explained by the metabolism of tryptophan into 4 indole, followed by further oxidization process in the liver, and ultimate decomposition to indirubin by bacteria (Hoessel et al., 1999). 1.1.3 Cellular absorption and metabolism So far only very limited evidence is available for the cellular uptake and metabolism of indirubins. One study in 2001 by Marko et al. determined the cellular content and distribution of three indirubin compounds (indirubin, I3M, and indirubin-5-sulfonate) through HPLC with UV detection (Marko et al., 2001). In LXFL529L cells treated by the respective compounds for 2 hr, the cellular content of indirubin and I3M (10 µM) did not differ significantly whereas indirubin-5-sulfonate was not detectable in the cells even with high concentration (50 μM). Of all the cellular content, 93 ± 6% of indirubin was localized in the particulate fraction whereas 43 ± 14% of I3M was detected in the cytosol. The highest growth inhibitory efficacy of I3M in the study above might be associated with its cytosolic distribution. Another report in 2004 by Guengerich et al. studied the metabolism of indirubins in vitro (Guengerich et al., 2004). Indirubin and I3M were incubated with liver microsomes prepared from rats that had been stimulated by corn oil or βNF (to activate cytochrome P450 enzyme) with or without NADPH. HPLC profiles showed that limited amount of indirubin was decomposed/metabolized into new products, an NADPH-dependent process. In contrast, almost all I3M were oxidized into product compound, which is also NADPH-dependent. 1.2 Pharmacological mechanisms of Indirubin and it derivatives 1.2.1 Anti-inflammatory activity 5 The anti-inflammatory activity of indirubin is mainly reflected by its anti-virus activity. Indirubin can inhibit viral replication or decrease viral yield. For example, I3M has been reported to inhibit Tat-induced replication of HIV-1 (primary and drug-resistance strains) RNA in human cell lines, in peripheral blood mononuclear cells and in macrophages through inhibition of CDK9 at low concentrations that did not affect cell proliferation (Heredia et al., 2005). I3M treatment can also significantly decrease cytomegalovirus yield in infected cells, attributed to its kinase inhibitory activity as well (Hertel et al., 2007). Besides, indirubin can modulate chemokine production in response to viral infection. In influenza virus-infected H292 human epithelial cells, indirubin inhibited the production of RANTES, a type of chemokine, at both the mRNA and protein level, although no mechanism was further investigated; in the same study, phosphorylation of NF-kB, its regulatory molecule IkBα and the p38 MAP kinase were also observed to be reduced after I3M treatment (Mak et al., 2004). In addition, indirubin can abrogate certain cellular abnormalities induced by viral protein as in the case of human papillomavirus infection, where I3M rescued the abnormal centriole duplication induced by one of the encoded viral proteins (Duensing et al., 2004). 1.2.2 Anti-cancer activity Indirubin was approved for clinical trials in China in the 1980’s (Institute of Haematology, Chinese Academy of Medicinal Sciences 1979). Indirubins have exhibited strong growth inhibitory effect on various human cancer cells, manifested by either cell cycle arrest (G2/M) (Hoessel et al., 1999; Yamaguchi et al., 2002) or cytotoxicity (mainly apoptosis) (Nam et al., 2005; Perabo et al., 2006; Ribas et al., 6 2006; Yamaguchi et al., 2002). An in vivo study carried out in rat tumor model provides further evidence for the anti-tumor activity of indirubins (Kim et al., 2007). 1.2.2.1 Clinical trials Indirubin was approved for clinical trials against chronic myelocytic leukaemia (CML) and chronic granulocytic leukaemia (Institute of Haematology, 1979). In one of these clinical trials, 26% of the 314 CML patients showed complete remission under indirubin treatment and 33% showed partial remission (Ma and Yao, 1983). Pharmacokinetics and pharmacodynamics data are not available. Minor toxicity was observed and mainly restricted to gastrointestinal tract, such as mild abdominal pain, diarrhea, nausea and vomiting; three cases of reversible pulmonary arterial hypertension and cardiac insufficiency were reported (Jiang, 1986). In a similar study, participating patients showed that indirubin had comparable efficiency as busulfan and there was no cross-resistance between the two tested compounds (Zhang, 1985). Before the approval for the clinical trials, long term toxicity test in dogs administering a high dose of synthetic indirubin (25 times of the dose for human application based on body weight) resulted in diarrhea and partial damage to the liver without interfering normal haematopoiesis, electroencephalogram activity and renal functions (Chang, 1985). 1.2.2.2 In vitro experimental evidence In vitro studies of growth inhibitory effect of indirubin and its derivatives mainly focused on two aspects in various human cancer cell lines: effects on cell cycle distribution and induction of cell death. 7 1.2.2.2.1 Cell cycle Progression through cell cycle is tightly regulated by internal and external signals (Morgan, 1997). Mitogenic factors can be the early signals that initiate entry into cell cycle, progression of which is the outcome of complex collaborations between various kinases and activator/inhibitor proteins (Eisenbrand et al., 2004; van den Heuvel and Harlow, 1993) (Fig. 1.2). Cyclin-dependent kinases (CDKs) play essential roles in cell cycle and they are activated by their binding partners, the cyclins; phosphorylation status affects the activities of various CDK/cyclin complexes (Morgan, 1997). Starting from the G0 phase of the cell cycle, cells go through G1 phase where active DNA synthesis occurs to prepare for the ensuing S-phase, which is primarily associated with cyclin D/CDK4- and cyclin D/CDK6-complexes. Cyclin D/CDK4 or 6 complexes hyperphosphorylate and inactivate the retinoblastoma protein (pRb), which induces release of E2F transcription factors from pRb (Harbour and Dean, 2000; Tamrakar et al., 2000). Consequently, E2F is activated and mediates transcription of proteins necessary for progression into S-phase, such as cyclin E, cyclinA, CDK1, and E2F-1 (Mueller, 2000). At an early stage of S-phase, CDK2 associates with cyclin E and later with cyclin A. CDK1/cyclin B and CDK1/cyclin A govern the G2 phase during which transition into M-phase and cell division are prepared (Sherr and Roberts, 1999). The CDK/cyclin complexes can be negatively regulated by small inhibitory proteins, also known as endogenous CDK inhibitors. Two families of CDK inhibitors are known: the CIP/KIP-family (p21CIP/WAF1, p27KIP1, p57KIP2), and the INK4-family (p15INK4b, p16INK4a, p18INK4c, p19INK4d/ARF). (Eisenbrand et al., 2004) Normal cell cycle progression is tightly controlled at distinct checkpoints. Many tumor cells lack appropriate checkpoint control, and deviations in cell cycle control are potentially present in all human tumors (DelSal et al., 1996). Mutations and/or 8 Figure 1.2 Cell cycle and major regulator proteins (Eisenbrand et al., 2004) 9 aberrant expression of important participants of the cell cycle have been identified, including pRb, cyclinD, CDK4, cyclinE, the INK4- and CIP/KIP-family, and many others (Eisenbrand et al., 2004). Therefore inhibition of cell cycle aberration is one promising target for cancer therapeutics and CDK inhibiting agents represent one focus in this direction. Several studies have shown indirubin can induce cell cycle arrest at G1/G0 or G2/M in a variety of human cancer cells, including chronic myeloid leukemic cell K-562, promyelocytic leukemia cells HL-60, mammary carcinoma MCF-7 cells, breast cancer cell MDA-MB-231, lung cancer cell A549, and breast carcinoma HBL-100, as well as a few rodent tumor cell lines (Hoessel et al., 1999). Most of these cell lines displayed G2/M arrest when treated by dose lower than 20 µM for 24 hr, and in some the cell cycle arrest was followed by apoptosis whereas in the case of HBL-100, the G2/M arrest was reversible after removal of I3M (Damiens et al., 2001). In MCF-7 and MDA-MB-231 cells, reduction of the G1/G0 population was also observed (Marko et al., 2001; Ribas et al., 2006). The mechanism responsible for cell cycle arrest induced by indirubin treatment has remained largely unknown. Preliminary data on the expression of cell cycle regulators do not match very well with each other, which might be explained by different cell systems and experimental approaches. For example, in HBL-100 cells treated by I3M (15 µM for 30 hr), no alterations in most regulators of the cell cycle based on ‘Cell cycle 1 GEArray’ or in mRNA and protein levels of Cyclin A, cyclin B1, and CDK1 were observed (Damiens et al., 2001), whereas in nocodazole synchronized MCF-7 cells treated by I3M after nocodazole release, time-dependent decrease was observed in protein levels of CDK1, CDK1/cyclin B complex, and cyclin B in complex with CDK1 (determined by immunoprecipitation using anti-CDK1), although total amount 10 of cyclin B increased (Marko et al., 2001). Another possible mechanism for cell cycle arrest induced by indirubins in tumor cells could be due to the inhibitory effect of indirubin on the kinase activity of CDK/cyclin complexes. For example, in I3M-induced G2/M-arrested MCF-7 cells, dose-dependent inhibition of CDK1 kinase activity was observed (Marko et al., 2001), consistent with the finding in HBL-100 cells (Damiens et al., 2001). Since indirubins are also effective inhibitors of CDK2/cyclinA and E, and of CDK4/cyclinD1, pRb phosphorylation in treated tumor cells can be a potential target of indirubin, blocking of which can prevent activation of E2F as a transcription factor, ultimately leading to G1/G0 arrest (Eisenbrand et al., 2004). Treatment of Jurkat cells with I3M reduced phosphorylation of pRB at Ser807/811 in a dose-dependent manner (Hoessel et al., 1999). Similar evidence can be found in LXFL-529L cells treated by another indirubin derivative, together with decreased phosphorylation of pRB at Ser780 and Ser795 (Eisenbrand et al., 2004). Ser780, Ser795 and Ser807/811 are all characteristic phosphorylation sites targeted by CyclinD/CDK4 (Merz et al., 2004). The evidence related to cell cycle interference fortifies the anti-tumor potential of indirubin derivatives although the mechanisms are not well understood. Development and selection of novel indirubin derivatives in vivo in human tumor xenograft models in nude mice has been reported (Eisenbrand et al., 2004). 1.2.2.2.2 Cytotoxicity Growth inhibition effects of Indirubin and its derivatives have been observed in various human cancer cells, as summarized in Table 1.1. 11 Table 1.1 Growth inhibitory effect of indirubin and its derivatives on various human cancer cell lines. IC50 values are labeled if available. Indirubin derivative Indirubin Indirubin5sulfonate I3M 5’-nitroindirubin monoxime 5’-fluoroindirubin monoxime 31.0 µM (Lee et al., 2005) >100 µM (Lee et al., 2005) 42.2 µM (Lee et al., 2005) >100 µM (Kim et al., 2007) >100 µM (Kim et al., 2007) 9.9 µM (Marko et al., 2001) 4.0 µM (Marko et al., 2001) >100 µM (Lee et al., 2005) >100 µM (Lee et al., 2005) >100 µM (Lee et al., 2005) 5.4 µM (Lee et al., 2005) 25.5 µM (Lee et al., 2005) 5.9 µM (Lee et al., 2005) 1.2 µM (Kim et al., 2007) 1.0 µM (Kim et al., 2007) 12.2 µM (Kim et al., 2007) 3.4 µM (Kim et al., 2007) 2.1 µM (Kim et al., 2007) 5.1 µM (Kim et al., 2007) 4.2 µM (Kim et al., 2007) 6.5 µM (Kim et al., 2007) 6.4 µM (Kim et al., 2007) >100 µM (Lee et al., 2005) >100 µM (Lee et al., 2005) 62.0 µM (Lee et al., 2005) >100 µM (Lee et al., 2005) 4.8 µM (Lee et al., 2005) >100 µM (Kim et al., 2007) 6.7 µM (Kim et al., 2007) >100 µM (Marko et al., 2001) >100 µM (Marko et al., 2001) (Damiens et al., 2001) >100 µM (Lee et al., 2005) (Hoessel et al., 1999) (KagialisGirard et al., 2007) (Perabo et al., 2006) 5’trimethyla cetaminoindirubin monoxime 6.4 µM (Kim et al., 2007) human cell lines A549 lung carcinoma Co12 Colon carcinoma HT-1080 fibrosarcoma SNU-638 stomach carcinoma PK3E-ras rat kidney epithelial cells LXFL 529L large cell lung carcinoma MCF-7 mammary carcinoma HBL-100 breast cancer HL-60 Myeloid leukemia Jurkat acute T cell leukemia lymphocyte stimulated by PHA RT4 transitional cell papilloma T24 transitional cell carcinoma TCCSUP transitional cell carcinoma 3.0 µM (Marko et al., 2001) 3.3 µM (Marko et al., 2001) 9.2 µM (Lee et al., 2005) (Perabo et al., 2006) (Perabo et al., 2006) 12 Indirubin and its derivatives induced apoptosis has been determined based on PARP cleavage (Nam et al., 2005), caspase activation (Kim et al., 2007), Annexin V and PI staining (Damiens et al., 2001; Kagialis-Girard et al., 2007), or chromatin ondensation visualized by DNA staining (Lee et al., 2005). But no detailed mechanistic study was carried out. Some studies investigated the Bcl-2 family protein changes; downregulation of anti-apoptotic member has been reported such as Mcl-1 and Survivin (Nam et al., 2005), although upregulation of Bcl-2 has also been reported (KagialisGirard et al., 2007). Cytochrome c release was demonstrated in 5’-nitroindirubinmonoxime treated A549 cells (Lee et al., 2005), suggesting the possible involvement of mitochondria. Besides apoptosis, necrosis is shown using Annexin V and PI staining (Damiens et al., 2001; Kagialis-Girard et al., 2007). In particular, I3M that is halogen substituted at the 7 position have been shown to induce caspase-independent cell death due to the lack of nuclear fragmentation typical of apoptosis and the insensitivity to caspase inhibitors (Ferandin et al., 2006; Ribas et al., 2006). Despite most studies that focus on the cytotoxic effects of indirubins, Xie et al in 2004 showed that I3M can prevent cerebellar granule neurons from apoptosis induced by potassium withdrawal through inhibition of c-Jun NH2-terminal kinase. Based on the available data, I3M display a differential toxicity between cancer cells and normal cells, which makes it a suitable candidate for cancer therapeutics. 1.2.2.2.3 Sensitization I3M has been reported to enhance the Tumor Necrosis Factor (TNF)-induced apoptosis through modulation of Nuclear Factor-κB (NFκB) signaling (Sethi et al., 2006). Without directly affecting the binding of NFκB to DNA, I3M suppressed TNF- 13 mediated NFκB activation in a dose- and time- dependent manner in human small cell lung carcinoma H1299 and human embryonic kidney A293 cells. I3M can also suppress NFκB activation induced by carcinogens and inflammatory stimuli, such as cigarette smoke condensate, H2O2, TNF, okadaic acid, and PMA, which are all potent activators of NFκB but through different mechanisms. In-depth studies showed that I3M blocked the phosphorylation and degradation of IκBα through the inhibition of IκBα kinase activation and phosphorylation and nuclear translocation of p65. In addition, I3M represses TNF-induced NFκB-dependent reporter gene expression; the following reporters have been tested: TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NFκB-inducing kinase, and IKKß. Other NFκB-regulated gene products that can be inhibited by I3M include proteins involved in anti-apoptosis, proliferation (cyclin D1 and c-Myc), and invasion (Fig. 1.3). I3M can also suppress cytokine-induced cellular invasion. The understanding of I3M as a potent inhibitor of NFκB fortifies its promises as a therapeutic agent. 1.2.2.2.4 Anti-angiogenesis A recent study reported that I3M had anti-angiogenic activity based on an automated, quantitative screening assay for anti-angiogenic compounds using transgenic zebra fish (Tran et al., 2007). I3M displayed dose-dependent anti-angiogenic activity in zebra fish with IC50 value of 0.31 µM, although it did not affect vasculogenic vessel development or established blood vessels. Furthermore, I3M has been observed to inhibit human umbilical vein endothelial cell tube formation and proliferation. Despite lacking detailed mechanistic study, the demonstrated anti-angiogenic activity of I3M in human endothelial cells enhances the potential of I3M as an anti-cancer agent. 14 Figure 1.3 A schematic representation of the effect of I3M on TNF-induced NFκB activation and apoptosis (Sethi et al., 2006) 15 1.2.2.3 In vivo Animal Model So far, only one rat tumor model has been established to assess indirubins’ antitumor activity in accessible published literature (Kim et al., 2007). A Korean group studied three novel indirubin derivatives (5-nitro-indirubinoxime, 5-fluoro-indirubinoxime, and 5-trimethylacetamino-indirubinoxime) both in vitro and in vivo. The experiment was designed as follows: RK3E-ras rat kidney epithelial cells harboring k-ras gene was subcutaneously inoculated on the left flank of three-week-old male Sprague-Dawley rats or alternatively injected into the oral mucosa. Treatment began on the 6th day after subcutaneous injection or 3rd day after oral injection, by direct injection of individual indirubin derivative into the tumor site every other day for a total of five times. It was found that indirubin derivatives showed potent antiproliferative activity on oncogenic RK3E-ras rat kidney cells, with IC50 ranging from 1 to 8 µM and caspase-7 activation followed by apoptosis was observed in these cells. Direct injection of indirubin derivatives significantly inhibited tumor growth in Sprague-Dawley rats with RK3Eras-induced solid and oral tumors, which was attributed to increased apoptosis and decreased cell proliferation based on histological observations. 1.2.3 1.2.3.1 Molecular targets of I3M Cyclin-dependent kinase (CDK) The most well characterized property of indirubin is its function as a CDK inhibitor. Known kinase targets of indirubin and its derivatives have been summarized in Table 1.2. Detailed structural study of the interaction between indirubin and CDK2 based on crystallography revealed that the kinase inhibitory effect of indirubin resulted from the high affinity binding of the molecule into the enzyme’s ATP binding site through van der Waals interactions and three hydrogen bonds (Hoessel et al., 1999). Similarly 16 Table 1.2 Kinase targets of indirubin and its derivatives. IC50 value for kinase inhibition is given if available. N.A.: not available (modified from Hoessel et al., 1999 and Leclerc et al., 2001). Indirubin 5-chloroindirubin I3M Indirubin-5sulphnic acid CDK1–cyclin B 10 0.4 0.18 0.055 CDK2–cyclin A 2.2 0.75 0.44 0.035 CDK2–cyclin E 7.5 0.55 0.250 0.15 CDK4–cyclin D1 12 6.5 3.33 0.3 CDK5–p35 5.5 0.8 0.1 0.065 Erk1 > 100 > 100 > 100 38 Erk2 43 > 100 > 100 > 100 c-Raf > 10 > 10 > 100 5.5 MAPKK > 100 > 100 > 100 3 c-Jun N-terminal Kinase > 100 21 N.A. 5.2 Protein kinase C α > 100 > 100 27 > 100 Protein kinase C ß 1 > 100 > 100 4 > 100 Protein kinase C ß 2 > 100 > 100 20 6.5 Protein kinase C γ > 100 > 100 8.4 > 100 Protein kinase C δ > 100 > 100 > 100 > 100 Protein kinase C ε > 100 > 100 20 > 100 Protein kinase C η > 100 > 100 52 > 100 Protein kinase C ζ > 100 > 100 > 100 > 100 > 1,000 600 6.3 > 1,000 > 1,000 380 9 480 Casein kinase 1 8.5 28 9 10 Casein kinase 2 Insulin- receptor tyrosine kinase > 100 > 100 12 1.5 > 1,000 550 11 320 c-Src tyrosine kinase 18 10 N.A. 3.8 c-Abl tyrosine kinase > 1,000 > 1,000 N.A. > 1,000 0.60 0.05 0.022 0.28 Enzyme cAMP-dependent protein kinase cGMP-dependent protein kinase GSK 3 17 inhibition of GSK-3ß by indirubin is achieved through its binding to the ATP pocket (Leclerc et al., 2001), suggesting a common interaction between indirubin and kinases. Detailed analyses of the interaction and possible molecular modifications for improved potency have been reviewed by Eisenbrand et al in 2004. The potent kinase inhibitory activities are responsible for some reported biological effects of indirubins. For example, the inhibition of CDK9 – the catalytic subunit of Positive transcription elongation factor b (P-TEFb) – by I3M contributes to the suppressed HIV-1 replication mediated by P-TEFb (Heredia et al., 2005). Indirubin compounds inhibit not only kinases but also phosphorylase. It is reported that indirubin-5-sulphonate inhibits glycogen phosphorylase with less extensive interaction comparing to that of CDK2 (Kosmopoulou et al, 2004). These evidences suggest that indirubin applications have a wide range of implications due to its enzyme inhibitory property. 1.2.3.2 Src kinase and Stat3 signalling pathway Stat3 signalling pathway has an important role in oncogenesis and therefore Stat3 protein is a promising anticancer target. Three indirubin derivatives have been shown to potently block constitutive Stat3 signalling in human breast cancer cells (MDA-MB468/-435) and prostate cancer cells (DU145). Mechanistic study revealed that the inhibition of Src kinase by indirubin derivatives led to decreased tyrosyl phosphorylation of Stat3 and thereby suppressed Stat3 DNA binding-activity, which ultimately affected the target genes of Stat3, such as the anti-apoptotic protein Mcl-1 and Survivin. (Nam et al., 2005) 1.2.3.3 Aryl hydrocarbon receptor (AhR) 18 Aryl hydrocarbon receptor (AhR), a member of the bHLH/PAS family of transcriptional factors, mediates the responses of some xenobiotics including indolecontaining compounds (Denison and Nagy, 2003). Upon ligand binding, AhR translocates from the cytoplasm to the nucleus and then binds to xenobiotic-responsive element, which stimulates the transcription of various genes including cytochrome P450 Cyp1A1 (Knockaert et al., 2004). The possibility of indirubins to be the physiological ligands of AhR was initially tested in yeast, and then proved in mammalian systems using in vitro and in vivo assays (Guengerich et al., 2004). Several studies provide evidences for a link between AhR activation and cell cycle control; using AhR-/- and +/+ cells and indirubin derivatives incapable of kinase inhibition, Knockaert et al (2004) suggested that AhR activation, rather than kinase inhibition, was responsible for the cytostatic effects of some indirubins. In contrast, the cytotoxicity of indirubins is irrelevant to AhR activation, but related to kinase inhibition. 1.2.3.4 Glycogen synthase kinase-3 (GSK3) Glycogen synthase kinase-3 (GSK3) plays critical roles in neuronal apoptosis and pathogenesis, especially neurodegenerative diseases. For example, GSK-3ß together with CDK5 is responsible for most abnormal Tau protein hyperphosphorylation in Alzheimer’s disease. Indirubins can bind to GSK-3ß’s ATP pocket and inhibit its kinase activity, which explains how I3M prevented tau phosphorylation in vitro and in vivo at sites of Alzheimer’s disease (Leclerc et al., 2001). In addition, I3M can inhibit GSK-3ß activation in MPTP-induced Parkinsonism models and prevent dopaminergic neurons from MPTP-induced neurotoxicity, including apoptosis, depletion of striatal dopamine and behavioral impairments (Wang et al., 2007). These evidence imply the 19 potential application of indirubin derivatives in the prevention of neurodegenerative diseases. 1.2.3.5 c-Jun NH2-terminal kinase (JNK) Kinase assay showed that I3M could inhibit all three isoforms of JNK in vitro. In cerebellar granule neurons, JNK activation induced by potassium withdrawal can be blocked by I3M in a dose-dependent manner; consequent inhibition of c-Jun phosphorylation correlated with reduced apoptosis. It is also postulated that I3M inhibits JNK through a mechanism different from its kinase inhibition, since other inhibitors of CDKs and GSK-3ß were ineffective in suppressing c-Jun phosphorylation (Xie et al., 2004). The evidence suggests the kinase inhibitory property of indirubin derivatives is still not fully understood. 1.2.3.6 Fibroblast growth factor receptor 1 (FGFR1) In cells stimulated by FGF-1, I3M inhibits autophosphorylation of FGFR1 and DNA synthesis, but it does not affect FGF-1 binding to the receptors or internalization of the ligand-receptor complex. Although the nature of the inhibition is still elusive, it is suggested that at low I3M concentration (5 µM) cell cycle is blocked by inhibiting the tyrosine kinase activity of FGFR1 whereas at higher concentration cell cycle interference by I3M is FGFR-independent. Furthermore, I3M inhibits the proliferation of FGFR-dependent cancer cells through inhibiting the tyrosine kinase activity of FGFR1 (Zhen et al., 2007). An interesting finding in Zhen’s study (2007) is that I3M, instead of inhibition, activates long-term p38 mitogen-activated protein kinase (p38 MAPK), an activity unrelated to the FGFR activity, which in turn stimulates extracellular signal-regulated 20 kinase 1/2 (ERK1/2). Evidence in this study also shows that I3M increased phosphrylated JNK, contrary to Xie’s study in 2004; the opposite results from two studies might due to different cells utilized: the former one used mouse embryonic fibroblast cell line (NIH/3T3) and the latter one used primary cerebellar neuronal cells from rats, suggesting cell type specific behavior of indirubin derivatives. 1.3 Apoptosis 1.3.1 General introduction Apoptosis, also termed as programmed cell death, is a conserved death pathway in mammals as well as lower organisms such as Caenorhabditis elegans and Drosophila melanogaster, required by multicellular organisms for regulating cell numbers or eliminating cells that are potentially detrimental or functionally useless (Twomey and McCarthy, 2005). Apoptosis was originally described based on morphological studies of developing vertebrate embryos, where isolated dying cells have the features of cell shrinkage, chromosomal condensation and cell fragmentation (Kerr et al., 1972). The elimination of cells by apoptosis is a fundamental event for successful embryonic development in terms of organogenesis and formation of complex multicellular tissues; apoptosis also functions in adult organisms to maintain tissue homeostasis (Danial and Korsmeyer, 2004), the critical role of which has been determined in genetic studies through the generation and functional characterization of overexpressing and/or deleting genes of the central apoptotic pathway in murine models (Colussi and Kumar, 1999; Hawkins and Vaux, 1997; Ranger et al., 2001; Yeh et al., 1999). Disturbance of the cellular homeostasis can be pathogenic: insufficient apoptosis can result in cancer or autoimmune diseases whereas overactive apoptotic events are observed in degenerative diseases or immunodeficiencies. 21 1.3.2 Apoptotic pathways The apoptotic machinery is widely conserved across species (Fig. 1.4). Studies of developmental cell death in C. elegans provided the first evidence for genes involved in apoptosis (Ellis and Horvitz, 1986), and led to the identification of several critical genes, such as ced-3. ICE (interleukin-1-ß-converting enzyme), initially identified as a novel mammalian protease responsible for processing pro-IL-1ß, was shown to be functional homologue of ced-3 in mammalian system (Yuan et al., 1993), suggesting that cystine proteases are essential components of apoptosis; homologues of the other apoptotic genes involved in cell death of C. elegans have also been identified and shown to be evolutionarily conserved (Chinnaiyan, 1999; Putcha and Johnson, 2004; Riedl and Shi, 2004). The mammalian homologues of ced-3 genes are defined as caspases (cystine-aspartate protease); 11 out of 14 identified so far are of human origin (Shi et al., 2002). The caspases that are involved in apoptosis are functionally subdivided into two categories: the initiator caspases and the effector caspases. Long pro-domains present in initiator caspases, death effector domain (DED) in pro-caspase 8 and 10 and caspase recruitment domain (CARD) in pro-caspase 2 and 9, are responsible for the oligomerization and autocatalytic activation of initiator caspases, which leads to the activation of effector caspases (pro-caspase 3, 6 and 7) by cleaving their short prodomain (Twomey and McCarthy, 2005). Downstream of effector caspase activation, cellular substrates in the cytoplasm or nucleus are cleaved, resulting the morphologic features of apoptosis (Degterev et al., 2003). There are primarily two apoptotic pathways in mammalian cells: extrinsic pathway initiating at the plasma membrane 22 upon ligation of death receptors and intrinsic pathway at mitochondria (Putcha et al., 2002; Wajant, 2002; Zimmermann et al., 2001) (Fig. 1.5). Figure 1.4 Evolutionarily conserved cell death pathways (Twomey and McCarthy, 2005) 23 Figure 1.5 Apoptotic pathways in mammalian system (Twomey and McCarthy, 2005). 24 1.3.2.1 Receptor-mediated apoptosis Activation of the death receptors results in caspase-8 activation and direct cleavage of downstream effector caspases such as caspase-3 (Vogler et al., 2007). Death receptors, members of the tumor necrosis factor (TNF) receptor gene superfamily, are distinguished from the other members of the superfamily by the presence of a cytoplasmic ‘death domain’, which is critical for the transmission of death signals from cell surface. There are several well characterized death receptors including Fas (Apo-1/ CD95), TNF receptor 1 (TNFR1), and TNF-related apoptosis-inducing ligandreceptor 1 or 2 (TRAIL-R1/DR4 or TRAIL-R2/DR5) (Walczak and Krammer, 2000); their corresponding ligands are Fas ligand (FasL), TNFα, and TRAIL. Signalling proteins interacting with death receptors possess a diverse set of modular protein motifs enabling homotypic interaction, including death effector domain (DED) and death domains (DD) (Itoh and Nagata, 1993) (Fig. 1.6). Fas, preassembled as a trimer, recruits adaptor FADD through DD domain, which in turn binds procaspase-8 via DED domain (Kischkel et al., 1995); together a signaling complex is formed on the cytoplasmic side of Fas, known as death-inducing signaling complex (DISC) (Muzio et al., 1996). High proximal concentration of caspase-8 is believed to induce autoproteolysis and subsequent proteolysis of caspase-3 and -7 (Scaffidi et al., 1998). DR4/5 activation leads to similar DISC complex formation and downstream events as the Fas pathway (Ashkenazi and Dixit, 1998). In the case of TNFR1, distinct complex formation occurs in a temporal pattern: within minutes of activation a TNFR1 complex (complex I) including TRADD, TRAF2, cIAP1, and RIP1 assembles at the plasma membrane to attract IKK and therefore activate the survival pathway of NF-kB; subsequent dissociation of complex I from TNFR1 can recruit FADD and procaspase8 to form complex II. When complex I-mediated NF-kB activation is 25 insufficient and inadequate cFLIP, an inhibitor of caspase-8, is expressed, complex II can induce apoptosis through caspase-8 activation (Micheau and Tschopp, 2003). Figure 1.6 Extrinsic death receptor pathways (Danial and Korsmeyer, 2004) 26 1.3.2.2 Mitochondrial-mediated apoptosis The intrinsic pathway of apoptosis is closely related to mitochondrial permeabilization of the outer membrane, induced by various cytotoxic stimuli and proapoptotic signaling molecules. The process is regulated by proteins from the Bcl-2 family, various functional protein components of the outer membrane, and mitochondrial lipids (Green and Kroemer, 2004). Upon permeabilization of the outer membrane, a group of proteins inside the intermembrane space is release to the cytosol, including cytochrome c, Smac/DIABLO, Omi/HtrA2, AIF and endonuclease G (Saelens et al., 2004). The release of cytochrome c induces the formation of the apoptosome complex containing cytochrome c, Apaf-1, and caspase-9. Cytochrome c binds to the Cterminal of Apaf-1, which facilitates the association of dATP with Apaf-1 and exposure of the N-terminal CARD domain. Initiator caspase-9 is then recruited and activated through the CARD-CARD domain interaction; subsequently, executioner caspase-3 is activated by caspase-9 and cleaves downstream substrates. In addition, membrane permeabilization can cause secondary events such as the drop of mitochondrial membrane potential (MMP) and deterioration of the normal function of complexes I and II, ultimately resulting in mitochondrial dysfunction and reactive oxygen species generation (Ricci et al., 2004). There is evidence suggesting a cytochrome c-/apoptosome-independent but Apaf1-dependent caspase activation (Fulda and Debatin, 2006). Other proteins released from the intermembrane space of mitochondria, such as Smac/DIABLO and Omi/HtrA2, promote caspase activation through antagonizing endogenous inhibitors of caspases – the inhibitor of apoptosis proteins (IAPs) (Fulda and Debatin, 2006). Omi/HtrA2 can also contribute to cell death by degrading XIAP, 27 cIAP1, cIAP2 and Apollon as a protease (Suzuki et al., 2004). Besides, AIF and endonuclease G translocate to nucleus upon release form mitochondria and contribute to chromosomal condensation and DNA fragmentation (Cande et al., 2004; Saelens et al., 2004). 1.3.2.3 Type I and type II cells For CD95 and TRAIL signaling pathway, two cell prototypes are distinguished. Type I cells activated sufficient caspase-8 through DISC complex for direct activation of the executioner caspase-3, whereas in type II cells, activated caspase-8 induced by death receptor is insufficient for full activation of effectors caspases and the mitochondrial participation is then required (Fulda et al., 2002). Cross talk between the extrinsic and intrinsic pathway can occur at different levels. In response to signals from death receptors, activated caspase-8 may cleave Bid, a BH3 domain only member of the Bcl-2 family, which subsequently translocates to mitochondria, and assists the release of cytochrome c, initiating the mitochondrial amplification loop (Cory and Adams, 2002). Furthermore, the activation of mitochondrial pathway, resulting caspase-6 activation, may feed back to the receptor pathway through cleavage of caspase-8 (Cowling and Downward, 2002). 1.3.3 Bcl-2 family Bcl-2 family proteins act as regulators of apoptosis through controlling the mitochondrial membrane permeability by pore-formation in the outer mitochondrial membrane (Tsujimoto, 2003). According to the structural and functional characteristics, they are categorized as anti-apoptotic members (Bcl-2, Bcl-XL, Mcl-1, 28 A1, and Bcl-w), multi-domain pro-apoptotic members (Bax, Bak) and BH3-only proapoptotic members (Bid, Bad, PUMA and NOXA)(Cory and Adams, 2002). The anti-apoptotic members display conservation in 4 conserved regions termed Bcl-2 homology (BH) 1-4 domains; structural study from Bcl- XL reveals that BH1, BH2 and BH3 domains form a hydrophobic pocket which is capable to accommodate a BH3 domain of a pro-apoptotic Bcl-2 member (Muchmore et al., 1996). The multi-domain pro-apoptotic members (Bax, Bak) shared BH1-3 domains, and an activation event is required to expose the hydrophobic portion of their BH3 domain for their interaction with Bcl-2 and Bcl-XL (Danial and Korsmeyer, 2004). Cells deficient for Bax and Bak proved resistant to most stimuli of the intrinsic pathway (Wei et al., 2001). In unstimulated cells, multi-domain Bax and Bak remain as monomers. Inactive Bax exists in the cytosol or is loosely attached to the mitochondrial membrane; upon receipt of a death signal, Bax undergoes conformational change and its N-terminal portion becomes exposed (transformed Bax) (Desagher et al., 1999). Bax and Bak then insert into the mitochondrial outer membrane as homo-multimers and ultimately lead to membrane permeabilization and release of proteins from intermembrane space (Danial and Korsmeyer, 2004). But the exact interaction of Bax and Bak and the precise mechanism by which proteins are released are still under investigation. Most BH3-only proteins function as death signal sensors which selectively respond to various apoptotic stimuli (Danial and Korsmeyer, 2004). Bid can be cleaved by caspase-8 and followed by N-myristoylation, which facilitates its translocation to the mitochondria. The intact BH3 domain of Bid can then trigger the oligomerization of Bax or Bak (Desagher et al., 1999). Bad is activated or deactivated by its phosphorylation status (Zha et al., 1996); PUMA and NOXA are transcriptionally regulated by p53 in response to DNA damage (Yu et al., 2001). Activation of BH3- 29 only proteins can directly or indirectly activate Bax and Bak whereas antiapoptotic proteins, such as Bcl-2 or Bcl-XL, can bind and sequester BH3-only proteins to prevent Bax and Bak activation and hence the amplification of the mitochondrial pathway. 1.3.4 Other regulatory mechanisms Other than Bcl-2 family members, IAPs (XIAP, cIAP1, cIAP2) also contribute to the regulation of apoptosis by modulating caspase activation. IAPs can inhibit caspase enzyme activity by direct interaction with the enzymatic domain or indirect blocking of substrate access (Riedl et al., 2001; Srinivasula et al., 2001). Therefore the degradation if IAPs can augment the activation of caspase cascade. 1.3.5 Apoptosis and cancer Disruption of the apoptotic pathways is one of the strategies cancer cells utilize to resist cell death. In response to death receptor ligand binding, the signaling process to induce apoptosis can be disturbed by impaired surface expression of CD95 or TRAIL receptors at different levels. One possible mechanism is through down-regulation or absence of death receptors on cell surface as in the case of drug-resistant leukemia or neuroblastoma cells where CD95 expression is strongly down-regulated (Friesen et al., 1997). Mutated CD95 genes have also been identified in various tumors (Debatin et al., 2003). Loss of functional TRAIL receptors (DR4 and DR5) expression (LeBlanc and Ashkenazi, 2003), deficient transport of DR4 and DR5 from intracellular protein synthesis stores (Jin et al., 2004), or the presence of decoy receptor for TRAIL (Sheikh et al., 1999), all can confer TRAIL resistance to cancer cells. Epigenetic alteration of death receptor genes, such as CpG-island hypermethylation and changes in chromatin 30 structure (Marks et al., 2001), can also contribute to the resistance to apoptosis in cancer cells. Moreover, interference to DISC complex formation has also been observed. Two variants of FLIP, a long form (FLIPL) and a short form (FLIPS), present in human cells have sequence homology with caspase-8 and caspase-10 but without catalytic activity (Krueger et al., 2001); therefore binding of FLIP to DISC complex instead of caspase-8/-10 forestalls caspase activation, which explains the high expression level of FLIP in many tumor cells resistant to CD95-/TRAIL- or druginduced apoptosis (Longley et al., 2006). PEA-15 is another protein that can interfere FADD and capase-8/-10 interaction (Hao et al., 2001). Disruptions to the intrinsic pathways are also very common in cancer cells. Overexpression of the anti-apoptotic Bcl-2 members is a dominant approach utilized by human follicular lymphoma (Tsujimoto et al., 1984); genetic mutations that inactivate the Bax gene have been observed in hematological malignancies as well as some solid tumors (Kitada et al., 2002; Rampino et al., 1997). Beside genetic mutations, deregulation of Bcl-2 family members can be due to transcriptional or posttranscriptional modification, such as regulation by NFκB (Cory and Adams, 2002). Decrease or absence of Apaf-1 activity has also been observed in a variety of tumors (Fulda and Debatin, 2006). Furthermore, mutations in some important tumor suppressor genes are crucial for cancer development since they can impinge on the apoptotic pathways (Brown and Attardi, 2005). For example, p53 mutation is most frequently detected in human cancers. Both death receptors (DR4 and DR5) (Liu et al., 2004; Pistritto et al., 2007) and pro-apoptotic BH-3 only proteins (PUMA, NOXA and Bid) (Oda et al., 2000; Sax et al., 2002; Yu et al., 2001) have been known to be transcriptionally regulated by activated p53. In addition, recent evidence suggests that p53 can directly promote 31 mitochondrial destabilization through binding with Bcl-2 and Bcl-XL, independent of its transcriptional activity (Mihara et al., 2003). 1.3.6 TRAIL signaling pathway as potential therapeutic target TRAIL is a promising candidate for clinical applications because it is relatively safe comparing to CD95L or TNFα (Walczak and Krammer, 2000), which is further supported by toxicity tests in non-human primates (Ashkenazi et al., 1999). TRAIL has also been proved for its efficacy to induce apoptosis in a wide spectrum of cancer cell lines; recombinant soluble TRAIL has been evaluated in phase I clinical trials (Fulda and Debatin, 2006). Despite the non-toxicity and prominent efficacy of TRAIL, TRAIL resistance occurs in certain tumors due to dominant anti-apoptotic signals; yet numerous studies have provided strong evidence for the sensitization of TRAIL-induced apoptosis by cytotoxic drugs or γ-irradiation. Various mechanisms have been identified in the context of a specific therapeutic combination and cell type, including transcriptional upregulation of the functional DR4 and DR5 (Meng and El-Deiry, 2001; Takimoto and El-Deiry, 2000), enhanced TRAIL receptor assembly (Lacour et al., 2004), downregulation of anti-apoptotic proteins including FLIP, Bcl-2 or Bcl-XL (Olsson et al., 2001), and up-regulation of pro-apoptotic proteins such as FADD or caspases (Micheau et al., 1999). Since many chemotherapeutics target on the intrinsic pathway, co-treatment with TRAIL can elicit both the mitochondrial and death receptor pathway, resulting in an augmented apoptotic response (Fulda and Debatin, 2006). Hence, TRAIL is a promising candidate that can be used in combination with existing chemotherapeutics. 32 1.4 Objectives of the study The main objective of this project is to study the molecular mechanisms involved in the anti-cancer effect of I3M, focusing on the apoptotic pathways involved. The specific aims of the study include the following: (1) To examine I3M-induced apoptosis in various human cancer cells (2) To determine the apoptotic pathway underlying I3M-induced apoptosis (3) To evaluate the critical role of Bcl-2 family members in I3M-induced apoptosis (4) To assess if I3M has any sensitization effect on TRAIL-induced cell death 33 Chapter Two Material and Methods 34 Chapter 2 Materials and Methods 2.1 Reagents, chemicals and plasmids Indirubin-3’-monoxime (I3M), 4’,6-diamidino-2-phenylindole (DAPI), thiazolyl blue tetrazolium bromide (MTT), cycloheximide (CHX) and TNFα were purchased from Sigma (St Louis, MO, USA). Propidium iodide (PI) was purchased from Molecular Probe (Eugene, OR, USA). Protease inhibitor cocktail was obtained from Roche (Mannheim, Germany). Pan caspase inhibitor z-VAD-FMK, caspase-8 inhibitor acIETD-CHO and caspase-9 inhibitor ac-LETD-CHO were purchased from Biomol (Plymouth meeting, PA, USA); caspase-3 inhibitor z-DEVD-FMK was from Calbiochem (San Diego, CA, USA). Other common chemicals were from Sigma. AntiPARP, anti-caspase-8, anti-bid, anti-caspase-9 (human specific), anti-caspase-3, and anti- COX IV antibodies were purchased from Cell signaling (Beverly, MA, USA), anti-Bax (N-20) polyclonal, anti-p53 (FL-393), and anti-p21 (C-19) antibody and goat anti-rabbit IgG HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anticytochrome c monoclonal antibody from Pharmingen (San Diego, CA, USA), anti-αtubulin monoclonal antibody from Sigma, and ImmunoPure® peroxidase conjugated goat anti-mouse IgG (H+L) from Pierce (Rockford, IL, USA). 2.2 Cell culture Human cervical cancer cell line HeLa, human hepatoma cell line HepG2, human colorectal cancer cell line HCT116, and human colorectal cancer cells HT29 were obtained from American Type Culture Collection (Manassas, VA) and human nasopharyngeal cancer cells CNE1 were obtained from Sun Yet-sat University (Guangzhou, China); they were maintained in Dulbecco’s modified Eagle’s medium 35 (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and antibiotics (Invitrogen, Carlsbad, CA, USA). Treatment details with I3M were illustrated in figure legends. All the chemical inhibitors were incubated 30 min before treatment. 2.3 Growth inhibition test: MTT MTT assay has been frequently used as an indication of growth inhibition (Hansen et al., 1989). Human cancer cells were seeded into 96-well plate 18 hr prior to various treatments; each treatment group was seeded in triplicate; a group of empty wells were used as blank control. At the end of the treatment, medium in each well was removed, and 25 µl of MTT (5mg/ml in sterilized PBS) was added. After 1 hr incubation at 37ºC with protection from light, 100 µl lysis buffer (50% DMF and15% SDS in dd-H2O, pH 4.6 ~ 4.7) was added into each well; the plates were shaked on an orbital shaker till all the crystal formed dissolved completely. The absorbance reading was recorded by a microplate reader Tecan® SPECTRAFLUOR PLUS (MTX Lab Systems, Inc.,Vienna) at the wavelength of 590 nm. 2.4 Western Blot Western blot analysis was performed as previously described (Zhou et al., 2006). Whole-cell lysate was prepared by lysing cells in M2 buffer (20 mM Tris, pH 7.4, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM ß-glycerol phosphate, 1 mM sodium vanadate and 1x protease inhibitor cocktail) and insoluble fractions were discarded after centrifugation at 15,000 g for 18 min. Equal amount of proteins were fractionated on SDS-PAGE gel in the Mini-PROTEAN II system (Bio-RAD) and 36 blotted onto PVDF membrane (Millipore). After blocking with 5% nonfat milk in TBST (10 mM Tris-HCl, 100 mM NaCl and 0.1% Tween-20), the membrane was probed with various primary antibodies followed by corresponding secondary antibodies, and then developed with enhanced chemiluminescence (Pierce) using a Kodak Image Station 440CF (Kodak). α-Tubulin was used as loading control. 2.5 Detection of apoptosis Human cancer cells were treated by I3M and then the apoptosis were detected using the following methods: (i) Morphological changes were observed under light microscope; and chromosomal condensation was detected by DAPI staining as previously described (Zhou et al., 2006). (ii) Percentage of the sub-G1 cells was measured by FACS Calibur (BD Biosciences, Heidelberg, Germany) using propidium iodide (PI) staining (Shen et al., 2000). (iii) PARP cleavage was detected in whole-cell lysate by western blotting. 2.5.1 DAPI staining After sucking out media gently, adhesion cells were washed once with 1x PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, and KH2PO4 2 mM in ddH2O, pH 7.4), then fixed with 70% ethanol (stored at -20ºC) for 2 min at room temperature. After being washed with 1x PBS gently, cells were stained by DAPI solution (3 µg/ml in PBS) for 5 min at room temperature. DAPI stock solution (6 mg/ml in DMSO) was kept at -20º C or -80ºC for long-term storage. Stained cells were washed again with 1x PBS gently and observed under fluorescent microscope. 37 2.5.2 PI staining followed by flow cytometry 106 to 107 Cells were collected and suspended in 1x PBS in a centrifuge tube. After centrifugation at 800 g for 5 min, cells were washed with 1x PBS and transferred to an eppendorf tube. Cells were resuspended in 50 µl of PBS and 450 µl of 70% ethanol (stored at -20ºC). Cells were fixed in ethanol for at least 2 hr. The ethanol-suspended cells were spun down by 800 g for 5 min and ethanol was decanted thoroughly. Cells were washed once with 1x PBS and suspended in 500 µl PI/Triton X-100 staining solution with RNase A (PI 20 µg/ml, Triton X-100 0.1%, and RNase A 0.2 mg/ml in PBS) for 15 min at 37ºC or 30 min at room temperature. Cells were then ready for analysis by FACS Calibur. 2.6 Measurement of caspase activity Caspase-3/7 activity was examined by Apo-One® Homogeneous Caspase-3/7 Assay (Promega, Madison, WI, USA) following manufacturer’s instruction. 100 µl of ApoOne® Caspase-3/7 reagent was added to each well of a 96-well plate containing 100 µl of blank, control or cells in culture. The contents of wells were mixed gently using a plate shaker at 300-500 rpm. The plate was then incubated at room temperature for1.5 hr (the incubation time can be from 30 min to 18 hr; the optimal incubation period is determined empirically). After incubation, the fluorescence intensity was measured at an excitation wavelength range of 485±20 nm and an emission wavelength range of 530±25 nm using Tecan SpectraFluor Plus (Tecan, Durham, NC, USA). 2.7 Measurement of surface expressions of death receptors Not more than one million HeLa cells, untreated or treated with I3M (20 μM×24 hr), were stained with Phycoerythrin (PE)-labled DR4 or DR5 (eBioscience, San Diego, 38 CA, USA) at room temperature for 30 min at dark. To control for nonspecific binding, PE-conjugated mouse IgG1, K isotype control were used as isotype-matched nonbinding antibodies. 20 μl of each of the specific antibodies or the isotype antibody together with 30 μl staining buffer was used for each sample. Cells were washed once with staining buffer before analysis by FACSCalibur using Cellquest software (BD). 2.8 Transfection 2.8.1 Transient transfection of siRNA HeLa cells were seeded 12-24 hr before the transfection in antibiotics-free medium and 30% confluency was achieved at the point of transfection. Negative control siRNA, and validated siRNA duplex targeting human Bid and Lamin A/C were purchased from Dharmacon. The cellular delivery of siRNA was carried out by using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions, optimized with various doses and post-transfection time, and evaluated by western blot. Cells were treated with I3M 72 hr after siRNA transfection, and apoptotic cell death was evaluated by PI staining followed by flow cytometry analysis. 2.8.2 Stable transfection of vector expressing Bid siRNA HeLa cells with stable knockdown of Bid, was generated using pSuper vector containing the following sequence: forward strand 5’ GATCCCC CCATAGAGGATGGTCTTAC TTCAAGAGA GTAAGACCATCCTCTATGG TTTTTGGAAA 3’; reverse strand 5’ AGCTTTTCCAAAAA CGATAGAGGATGGTCTTAC TCTCTTGAA TAACCATTCGTGGGTGGTC GGG 3’, which carries a neomycin selection marker (a gift from Dr. Song ZW, BTI, A*Star). Corresponding control HeLa cells expressing empty pSuper vector with 39 neomycin selection marker were also generated. After transient transfection of the above pSuper vectors, cells that survived two weeks of selection were used to generate single cell clones by limiting dilution. G418 Sulfate (Promega) 500 µg/ml was applied in the complete DMEM medium during selection and after selection, but not during any treatment. 2.8.3 Detection of Bcl-2 or CrmA transfected cells using flow cytometry HeLa cells were seeded 12 hr before the transfection in antibiotics-free medium and 90% confluency was achieved at the point of transfection. Cells were transfected with pcDNA3.1 empty vector, Bcl-2 expression vector or CrmA expression vector (a kind gift from Dr. ZG Liu, NCI, USA) using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions; pmaxGFP vector (Amaxa Inc., Gaithersburg, MD, USA) was co-transfected as a transfection marker and only successful transfected cells were analyzed as described before (Won et al., 2005). Briefly speaking, only cells expressing GFP, as detected by FACSCalibur, were analyzed for their DNA content and presented in the data. The voltage used was determined by control samples with or without GFP expression. After 48 hr of transfection, the cells were treated with 20 µM I3M for 24 hr. Cell death was determined by percentage of sub-G1 events and morphological changes examined under inverted fluorescent microscope (Nikon, Tokyo, Japan). 2.9 Detection of Bax conformational change 2.9.1 Immunofluorescence HeLa cells were seeded in 8-well chamber slides (Nalge Nunc, Rochester, NY, USA) 24 hr before treatment. After treatment cells were fixed in 3% paraformaldehyde for 40 30 min at room temperature and permeablized for 2 min with 0.2% CHAPS in PBS. After blocking with 1x PBS containing 0.2% Tween-20, 5% FBS and 3% BSA for 1 hr, cells were incubated with anti-Bax 6A7 antibody overnight at 4ºC. After washing with PBS containing 0.2% Tween-20, cells were incubated with anti-mouse Alexa 633 secondary antibody for another 1 hr at room temperature. Coverslips were mounted onto slides using ProLong anti-fade mounting reagent (Molecular Probes). Cells were then visualized under Olympus FLOVIEW V500 confocal microscope with 60x oil lens. 2.9.2 Immunoprecipitation Immunoprecipitation was carried out as previously described (Zhou et al., 2006) with minor changes. Briefly, cells were lysed in CHAPS lysis buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 1% CHAPS) containing protease inhibitors (Yamaguchi et al., 2002). The cell lysates were normalized for protein content and pre-cleared by incubating 500 µg of total protein with 50 µl of protein G-agrose for 60 min on ice. After spinning at 10,000g for 10 min, pre-cleared lysate (supernatant) were transferred to a new eppendorf tube and incubated with 2 µg of anti-Bax 6A7 monoclonal antibody (Sigma) in 500 µl of CHAPS lysis buffer overnight at 4ºC. Afterwards, 25 µl of protein G-agrose were added and incubated for additional 3 hr at 4ºC. Protein Gagrose beads were washed carefully in CHAPS lysis buffer for 3 times and then boiled in loading buffer containing 5% ß-mercaptoethanol. Conformational changed Bax in the immunoprecipitates were detected by western blot using anti-Bax polyclonal antibody. 41 2.10 Subcellular fractionation Whole-cell lysate was prepared using M2 buffer as described above. Cytosolic and nuclear fractions were obtained according to a protocol previously described with minor modifications (Zhou et al., 2006). In brief, sample cells were harvested and centrifuged at 1,500 rpm for 5 min. The pellet was washed with ice-cold PBS and resuspended in isotonic homogenization buffer (10 mM Tris-HCl, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM PMSF, 1 mM DTT, and 1x protease inhibitor cocktail, pH 6.7); cells were incubated on ice for 10 min and homogenized by passing through a syringe with gauge #27 needle for 15 min at 4ºC. The lysate was then subject to a series of centrifugation at 50 g for 10min, 1,200 g for 5min, 12,500 g for 20 min to fractionate intact cells, nuclei and mitochondria, respectively. The supernatant generated after a final centrifugation of 60,000 g for 30 min was collected as the cytosolic fraction. The mitochondria pellet was dissolved in M2 buffer and the soluble lysate was taken as the mitochondrial fraction. 2.11 Long-term clonogenic assay 5000 cells are seeded into each well of a 6-well plate 24 hr before treatment. After designated treatment, treatment medium is changed to complete medium and cells are allowed to grow for about 1 week or longer depending on cellular growth rate. Then cells were fixed by 3% paraformaldehyde for 30 min at room temperature and stained by 1% crystal violet for 1 hr at room temperature. After 3 times wash with 1x PBS, the 6-well plate is photographed with digital camera after complete drying. 2.12 Statistical analysis 42 All numerical data were presented as mean ± S.D. of at least three independent experiments. Statistical significance was assessed by Student’s t-tests (two-tailed distribution, two-sample unequal variance). P values less than 0.05 were considered significant. 43 CHAPTER THREE RESULTS 44 Chapter 3 Results 3.1 I3M induces apoptosis in a time- and dose-dependent manner in human cancer cells With I3M treatment, we observed the characteristics of apoptosis – membrane blebbing, chromosomal condensation and DNA fragmentation in HeLa, HepG2 and HCT116 (Fig. 3.1). Growth inhibitory effect of I3M on the cancer cells was measured using MTT assay (Fig. 3.2). I3M-induced apoptosis was quantified using sub-G1 analysis; we observed a time- and dose-dependent manner in the three cancer cells (Fig. 3.3A & B). Among them, HeLa cells are most susceptible to I3M. In addition, PARP cleavage, another hallmark of apoptosis, was also detected in HeLa cells in a similar time- and dose-dependent pattern (Fig. 3.3C). Similar results were observed in HepG2 and HCT116 cells. 3.2 I3M leads to caspase activation To understand the apoptotic machinery involved in I3M-induce apoptosis, we examined the caspase activation. Evident caspase-8 cleavage commenced at 12 hr and almost all were cleaved at 24 hr (Fig. 3.4A). Cleavage of caspase-9 and caspase-3 was also detected in a similar temporal pattern (Fig. 3.4A). In addition, we quantified the activity of effector caspases (caspase-3/7) in the three cancer cells and found that the degree of activity (Fig. 3.4B) corresponded to that of apoptosis detected by sub-G1 analysis (Fig. 3.3A). To confirm the involvement of the above-mentioned caspases, we utilized various synthetic caspase inhibitors to test their protective effects on I3M-induced cell death. 45 Figure 3.1 Morphological changes and nuclear condensation of cancer cells in response to I3M treatment. HeLa (A), HepG2 (B), and HCT116 (C) were treated by I3M 20 μM for 24 hr. Representative images were captured under an inverted fluorescent microscope after DAPI staining. 46 Figure 3.2 Growth inhibitory effect of I3M on cancer cells. MTT reduction was observed in HeLa, HepG2 and HCT 116 in a dose-dependent manner when cells were treated by the indicated concentrations of I3M for 24 hr. Data are presented as means ± S.D. from at least three independent experiments. 47 A. B. C. 48 Figure 3.3 Time- and dose-dependent apoptosis in cancer cell lines. A. Time- dependent responses to I3M treatment. The percentage of sub-G1 events in HeLa, HepG2 and HCT 116 cells treated with 20 μM I3M for the indicated time periods. Inserted are typical histograms derived from flow cytometry analysis of DNA content in control and I3M-treated HeLa cells (20 μM×24 hr). B. Dose-dependent responses to I3M treatment. The percentage of sub-G1 events in HeLa, HepG2 and HCT 116 cells were determined when treated with different concentrations of I3M for 24 hr. C. I3Minduced PARP cleavage in HeLa cells determined by western blot. Data in panel A and B are presented as means ± S.D. from at least three independent experiments. 49 A. B. Figure 3.4 I3M-induced caspase activation. A. I3M-induced caspase-8, -9 and -3 cleavages in HeLa cells determined by western blot. B. I3M-induced caspase 3/7 activity measured by Apo-OneTM Caspase-3/-7 Assay Kit. Three type of cancer cells were treated by 20 μM I3M for the indicated time periods. Data are presented as means ± S.D. from at least three independent experiments. 50 Pretreatment with a pan caspase inhibitor (z-VAD) or a caspase-3 inhibitor (z-DEVD) completely protected I3M-induced apoptosis (Fig. 3.5). In contrast, pretreatment with a caspase-8 inhibitor (ac-IETD) or a caspase-9 inhibitor (ac-LEHD) only partially protected apoptosis induced by I3M (Fig. 3.5). Correspondingly, the pan caspase inhibitor (z-VAD) and the caspase-3 inhibitor (z-DEVD) suppressed the effector caspase activity to the basal level, whereas the caspase-8 and caspase-9 inhibitors (AcIETD and Ac-LEHD) only partially inhibited the effector caspase activity (Fig. 3.6). Data from Fig. 3.4, Fig.3.5 and Fig. 3.6 collectively suggest that I3M-induced apoptosis involves both the extrinsic and intrinsic apoptotic pathways. 3.3 I3M induces increased surface expression of death receptors accompanying p53 up-regulation To provide a possible mechanism for the activation of the extrinsic pathway induced by I3M, we first evaluated the surface expression of the death receptor 4 and 5 (DR4 and DR5) in HeLa cells. Upon treatment with I3M for 9 hr, levels of both receptors increased significantly (Fig. 3.7A). Such observations were confirmed by the total protein level of DR4 and DR5 determined by western blot (Fig. 3.7B). It has been reported that the expression of DR4 and DR5 is transcriptionally regulated by tumor suppressor gene p53 (Liu et al., 2004; Pistritto et al., 2007). Here we also observed a time-dependent increase (with a peak at 12 hr) of the p53 protein level in cells treated with I3M (Fig. 3.7C). The concurrent increase of the p21 protein level indicated the transcriptional activation of p53 induced by I3M in HeLa cells (Fig. 3.7C). 51 Figure 3.5 Inhibition of I3M-induced apoptosis by synthetic caspase inhibitors. Protective effects of inhibitors were measured by percentage of sub-G1 events. HeLa cells were treated by 20 μM I3M together with 50 μM individual caspase inhibitor for 24 hr. Each treatment is represented by a typical histogram showing the sub-G1 peak; and the inserted image showing the cell morphology under light microscopy. The numerical data indicates the percentage of sub-G1 cells. Data are presented as means ± S.D. from at least three independent experiments. 52 Figure 3.6 Inhibition of I3M-induced caspase activation by synthetic caspase inhibitors. Caspase 3/7 activity was measured by Apo-OneTM Caspase-3/-7 Assay Kit. HeLa cells were treated with 20 μM I3M together with 50 μM individual caspase inhibitor for 12 hr. Data are presented as means ± S.D. from at least three independent experiments. 53 A. B. C. Figure 3.7 I3M-induced enhanced surface expressions of DR4 and DR5. A. Surface expression of DR4 and DR5. Untreated HeLa cells (Ctrl) and HeLa cells upon 9 hr of exposure to I3M (20 μM) were analyzed by FACS using PE-conjugated DR4 and DR5 antibodies. Isotype-matched antibodies (Iso) were used to control for unspecific binding. B. Total protein level for DR4 and DR5 determined by western blot. The cells were treated with I3M as indicated. C. I3M-induced p53 and p21 upregulation. HeLa cells were treated as indicated and protein levels were determined by western blot. 54 3.4 I3M causes Bid cleavage The extrinsic death receptor pathway can initiate the mitochondrial amplification loop in type II cells by caspase-8 mediated Bid cleavage and subsequent translocation of tBid (truncated Bid) to the mitochondria (Li et al., 1998; Luo et al., 1998). In this study, since I3M-induced apoptosis involves both caspase-8 and -9 activation (Fig. 3.4A), we thus examined whether I3M could induce Bid cleavage. I3M led to evident Bid cleavage in a time- and dose-dependent manner (Fig. 3.8A), which is completely prevented by a pan-caspase inhibitor (z-VAD) or a caspase-8 inhibitor (Ac-IETD) (Fig. 3.8B), in correspondence with the pattern of protection regarding cell death (Fig. 3.5). In order to confirm the role of Bid in I3M-induced apoptosis, we managed to knockdown the Bid expression levels in HeLa cells using the siRNA technique. With transient transfection, about 20% of protection can be observed in cells transfected with Bid SiRNA comparing to cells transfected with non-silencing (negative control) or Lamin A/C (positive control) siRNA (Fig. 3.9B); the suppression of Bid expression was proved by western blot (Fig. 3.9A). Furthermore, we established the stable Bid knockdown HeLa cells using the pSuper vector system (Fig. 3.10A). In HeLa cells with Bid stable knonckdown, there is a 50% reduction for the percentage of apoptosis induced by I3M as determined by sub-G1 analysis (Fig. 3.10B). Consistently, PARP cleavage was also partially salvaged comparing to the cells expressing the control vector (Fig. 3.10C). 55 A. B. Figure 3.8 Bid cleavage in response to caspase activation in I3M-induced apoptosis. A. I3M-induced Bid cleavage in a time- and dose-dependent manner in HeLa cells under the indicated treatment conditions. B. Caspase inhibitors blocked I3M-induced Bid cleavage. HeLa cells were treated with 20 μM I3M for 24 hr with the absence or presence of 50 μM individual caspase inhibitor. 56 A. B. Figure 3.9 Protection conveyed by Bid siRNA transient transfection against I3M- induced apoptosis. A. Reduction of Bid protein level in HeLa cells transiently transfected with Bid siRNA; non-silencing siRNA (non-sil) was used as negative control and validated Lamin A/C siRNA as positive control. B. Protection against I3M-induced apoptosis in HeLa cells transfected with Bid siRNA. Cells were treated with I3M (20 μM×24 hr) and apoptosis was measured by percentage of sub-G1 events. Data are presented as means ± S.D. from three independent experiments. 57 A. B. C. Figure 3.10 Protection conveyed by Bid knockdown against I3M-induced apoptosis. A. Reduction of Bid protein level in HeLa cells with stable expression of the pSuper Bid SiRNA vector in comparison to cells with the control vector. B. Protection against I3M-induced apoptosis in HeLa cells stably expressing Bid SiRNA. Cells were treated with I3M (20 μM×24 hr) and apoptosis was measured by percentage of sub-G1 events. Data are presented as means ± S.D. from three independent experiments. Statistical significance was analyzed by Student’s t-test (**: p < 0.01 when compared with the control group). C. Bid knockdown partially protected I3M-induced PARP cleavage. HeLa cells stably expressing either the control vector or Bid siRNA were treated with I3M (20 μM×24 hr). PARP cleavage was determined as Fig. 3.3. 58 3.5 I3M induces Bax conformational changes In response to Bid or other BH3-only proteins, multi-domain pro-apoptotic Bcl-2 family members, such as Bax and Bak, can be conformationally activated to form homo-multimers/complex in the mitochondrial membrane and thereby increase the membrane permeability (Tsujimoto, 2003). Here we investigated the conformational change of Bax using the following two methods: (i) immunofluorescence detected using a specific antibody (anti-Bax 6A7) that can recognize the N-terminal of the transformed Bax (Hsu and Youle, 1998; Nechushtan et al., 1999), and (ii) immunoprecipiation and western blot. In I3M-treated HeLa cells, there is a time- and dose-dependent increase of red fluorescence (Fig. 3.11A), indicating the increased amount of transformed Bax. Such results are consistent with the immunoprecipiation data in Fig. 3.11B that there is a time- and dose-dependent increase of Bax pulled down by anti-Bax 6A7. Bands at about 42 kDa were observed in Fig. 3.11B and suspected to be the dimmer form of Bax. Furthermore, Bax conformational change was caspase-dependent as both a pan caspase inhibitor (z-VAD) and a caspase-8 inhibitor (ac-IETD) significantly blocked such changes (Fig. 3.11C). Finally, Bid knockdown also significantly suppressed Bax conformational changes induced by I3M (Fig. 3.11D), suggesting that that Bax acts downstream of Bid in I3M-induced apoptosis. 59 A. B. C. 60 D. Figure 3.11 Bax conformational change following caspase-8 activation in I3M- treated HeLa cells. A. Conformational changes of Bax detected by immunofluorescence staining and shown in red fluorescence. Cell nuclei were counterstained by DAPI. Representative images were captured under a confocal microscope. B. Conformational changes of Bax detected by immunoprecipitation (IP) and western blot (WB). Following designated treatments, transformed Bax was immunoprecipitated using anti-Bax 6A7 antibody and then subject to western blot analysis using anti-Bax N-20 antibody. C. Caspase-dependent Bax conformational change. HeLa cells were treated with I3M (20 μM) with the presence of caspase inhibitors (50 μM) for 24 hr and the transformed Bax was detected as in Panel A. D. Reduced Bax conformational change in HeLa cells stably expressing Bid siRNA. Cells 61 were treated with 20 μM I3M for 24 hr and the transformed Bax was detected as in Panel A. 62 3.6 I3M induces cytochrome c release Increased mitochondrial membrane permeability due to Bax-Bak complex formation can cause the release of cytochrome c and other apoptogenic factors from the mitochondrial intermembrane space, which would eventually stimulate caspase-9 and caspase-3 activation and execute apoptosis (Saelens et al., 2004). To furtherinvestigate I3M’s effect on mitochondria, we examined cytochrome c release from mitochondria. A time-dependent increase of cytochrome c in the cytosolic fraction was observed (Fig. 3.12). 3.7 Overexpression of Bcl-2 or CrmA partially blocks I3M-induced apoptosis Data presented above highlight the critical role of the pro-apoptotic Bcl-2 family members in I3M-induced apoptosis at the site of mitochondria. Here we used genetic approaches to further examine the role of the anti-apoptotic Bcl-2 protein in I3Minduced apoptosis. HeLa cells were transiently transfected with expression vector of either Bcl-2 protein or the viral protein cytokine response modifier A (CrmA), a known specific caspase-8 inhibitor (Miura et al., 1995), together with a green fluorescent protein (GFP) construct as a transfection marker. For a more reliable analysis of the effects of overexpressed Bcl-2 or CrmA on I3M-induced apoptosis, we analyzed the DNA content/sub-G1 profile only among the transfected cell population, indicated by the expression of GFP (Fig. 3.13A). Based on the morphological changes of transfected cells (with green fluorescence) (Fig. 3.13B) and flow cytometry analysis of those transfected cells (Fig. 3.13C), overexpression of CrmA or Bcl-2 provided strong protection against I3M-induced cell death (Fig. 3.13D). 63 Figure 3.12 I3M-induced cytochrome c release. HeLa cells were treated by 20 µM I3M for 0, 6, 12, 18 and 24 hr and subjected to subcellular fractionation to isolate the cytosolic fractions. Protein levels were examined by western blot. Cox IV indicated the cytosolic fractions were clear of mitochondria and tubulin was used as a loading control. 64 A. B. 65 C. D. Figure 3.13 Ectopic expression of Bcl-2 or CrmA protects against I3M-induced apoptosis. A. HeLa cells were first transiently transfected with pcDNA empty vector, 66 Bcl-2, or CrmA expression vector, respectively, together with PmaxGFP as a transfection marker. After treatment, cells were collected and stained with PI for DNA content analysis. Among total of 20,000 cells from each group analyzed using flow cytometry, only those transfected cells with GFP expression and PI staining were presented for the analysis of sub-G1 events. B. Morphology of transiently transfected HeLa cells viewed under fluorescent microscope; 48 hr post transfection, cells were treated by I3M (20 μM×24 hr) as indicated. Successfully transfected cells were indicated by green fluorescence. C. Representative histograms from each transfection group with or without I3M treatment. D. Quantification of percentage of sub-G1 events. Data are presented as means ± S.D. from three independent experiments. Statistical significance was analyzed by Student’s t-test (**: p < 0.01 when compared with the pcDNA group; *: p < 0.05). 67 3.8 I3M sensitizes cancer cells to TRAIL–induced apoptosis. It has been shown that I3M was able to sensitize TNF-induced apoptosis in human cancer cells (Sethi et al., 2006). Here we further assessed the effect of I3M on TRAILinduced cell death. First, we tested the cytotoxicity of TRAIL on human cancer cell lines originated from various tissues, including human cervical cancer cells HeLa, human nasopharyngeal cancer cells CNE1, human live cancer cells HepG2, and human colorectal cancer cells HT29. Some cancer cells were reported to be TRAIL resistant (Shi et al., 2005). According to our data, up to as high as 200 ng/mL TRAIL induced less than 50% of apoptosis in CNE1, HepG2, or HT29 cells even after 24 hr treatment (Fig. 3.14A), while the HeLa cells tested in this experiment were more sensitive to TRAIL-induced apoptosis. When these cells were pretreated with I3M for 2 hr, followed by a subcytotoxic concentration of TRAIL for additional 6 hr, apoptosis in all four cell lines investigated increased, as measured by percentage of sub-G1 events, whereas I3M or TRAIL alone did not induce significant cell death (Fig. 3.14B). Fig. 3.14C shows the chromatin condensation in HeLa cells treated with I3M and TRAIL. These data suggest that I3M pretreatment not only markedly sensitizes TRAILresistant cancer cells but also significantly accelerates the cell death process. Longterm effects of I3M and TRAIL were tested in these cancer cells using colony formation assay. I3M (10 µM) alone has a certain degree of growth inhibitory effects on HeLa cells in the long term (Fig. 3.14D) whereas it was not cytotoxic in the shortterm apoptosis assay (Fig. 3.14B). A combination of I3M and TRAIL completely suppressed HeLa cell growth and colony formation (Fig. 3.14D); similar results were observed in the other three cancer cell lines. 68 A. B. 69 C. D. Figure 3.14 I3M sensitizes human cancer cells to TRAIL-induced apoptosis. A. HeLa, CNE1, HepG2, and HT29 cells were treated with various concentrations of TRAIL for 24 hr. B. Cells were first pretreated with indicated concentrations of I3M for 2 hr, followed by treatment with a subtoxic concentration of TRAIL for another 6 hr (0.5 ng/ml for HeLa, 1 ng/ml for CNE1, 5 ng/ml for HepG2 and H T29). A and B, at the end of treatment, apoptosis was measured by percentage of sub-G1 events. Data are presented as means ± S.D. from three independent experiments. C. Apoptotic morphologic changes in HeLa cells with combined treatment with I3M (10 µM×8 hr) and TRAIL (1 ng/ml×6 hr). Top, cells observed under a normal light microscope; 70 bottom, cells with DAPI staining under an inverted fluorescence microscope. D. Colony formation assay. HeLa cells were plated on six-well plates (5,000 cells/well) and treated with I3M alone (10 µM), TRAIL alone (1 ng/ml), or their combination for 1 week. The survival clones were stained with 0.5% crystal violet. 71 CHAPTER FOUR DISCUSSIONS AND CONCLUSIONS 72 Chapter 5 Discussions and conclusions Previous studies have demonstrated that indirubin and its derivatives are promising anti-cancer agents based on the following observations: (i) they are capable of selectively inducing apoptotic cell death in a wide spectrum of human cancer cells with minimal toxicity on normal cells (Kim et al., 2007; Nam et al., 2005; Perabo et al., 2006; Ribas et al., 2006); and (ii) in vivo study in rat model has proved their efficacy in arresting tumor growth (Kim et al., 2007). However, the molecular mechanisms underlying the apoptotic cell death induced by indirubin and its derivatives have not been fully elucidated. In this study we provide convincing evidence demonstrating that I3M-induced apoptosis engages the extrinsic death receptor pathway with a type II-cell behavior in which the pro-apoptotic bcl-2 family members Bid and Bax play a critical role. In addition, we prove I3M can enhance TRAIL-induced apoptosis in human cancer cells. 4.1 I3M induced apoptosis in HeLa, HepG2 and HCT116 Growth inhibitory effects of indirubin have been proven in various human cancer cells with evidence pointing to apoptosis. In our study, observed chromatin condensation based on DAPI staining (Fig. 3.1), DNA fragmentation detected by flow cytometry (Fig. 3.3A & B), PARP cleavage (Fig. 3.3C), and caspase cleavage and activation (Fig. 3.4) provide convincing evidence for the occurrence of apoptosis in HeLa, HepG2 and HCT116 cells. Among the three cell lines, HeLa is most sensitive to I3M whereas HCT116 is least. A previous study has shown that another colon carcinoma, Co12, is not sensitive to I3M with IC50 value greater than 100 µM (Lee et al., 2005), suggesting 73 that I3M is not suitable for the treatment of colon cancer although other indirubin derivative has shown a much better efficacy (Table 1.1). Despite that most previous studies conclude that indirubins induce apoptosis in cancer cells, necrosis has also been reported based on Annexin V and PI co-staining (Damiens et al., 2001; Kagialis-Girard et al., 2007). Furthermore, Ribas et al. (2006) reported caspase-independent cell death induced by 7-substituted indirubins, which has a marginal inhibitory effect towards CDKs and GSK-3 comparing to other indirubin derivatives. Therefore the chemical structure of indirubins influences the type of cell death induced and the inhibition of CDKs and GSK-3 seems to contribute to the engagement of the apoptotic pathway. In the present study, the cell death induced by I3M in HeLa cells can be completely protected by the pan caspase inhibitor z-VAD (Fig. 3.5), excluding the possibility of other forms of cell death. 4.2 Apoptosis induced by I3M recruits extrinsic pathways with type II response Although existing study suggests kinase inhibition might be responsible for the cytotoxic effect (Knockaert et al., 2004), the molecular targets of indirubins as well as signaling process upstream of apoptotic commitment are still unidentified. Our study is the first to prove the involvement of the extrinsic death receptor pathway in I3Minduced apoptosis, as demonstrated by evident caspase-8 activation at early time points (Fig. 3.4B), and the protective effect of a synthetic caspase 8 inhibitor (Fig. 3.5), as well as overexpression of a viral caspase 8 inhibitor CrmA (Fig. 3.13). Similar mechanism of action has been reported for a number of other natural products. For example, andrographolide, an extract from a traditional herbal medicine Andrographis paniculata, has been shown to induce apoptosis in HepG2 cells via caspase-8 74 activation (Zhou et al., 2006). Similarly, prodelphinidin B-2,3,3’-di-gallate from Myrica rubra (Kuo et al., 2004) and the water extract of Phyllanthus urinaria have been shown to trigger apoptosis via the Fas/FasL system (Huang et al., 2004). Furthermore, we observed increased surface expressions, as well as total protein level, of both death receptor DR4 and DR5 in HeLa cells upon I3M treatment (Fig. 3.7A & B). DR4 and DR5, also known as TRAIL-R1 and TRAIL-R2, respectively, contain functional cytoplasmic DD motifs, which associate with FADD upon activation by apoptotic signals such as TRAIL (Cretney et al., 2007; Takeda et al., 2007). FADD contains the DED and is involved in the activation of caspase-8 (Voortman et al., 2007). Therefore, increased surface expression of DR4 and DR5 observed in I3Mtreated cells (Fig. 3.7A) might contribute to the caspase-8 activation observed in Fig. 3.4A. Yet, we did not investigate the activation of TNFR1 or Fas in our current study, which can also lead to the activation of caspase-8. It has been reported that expression of DR4 or DR5 is transcriptionally regulated by p53 tumor suppressor gene (Liu et al., 2004; Pistritto et al., 2007). In this study, the significantly elevated p53 and p21 protein level in I3M-treated cells (Fig. 3.7B) suggests the possibility that I3M promotes DR4 and DR5 expression via activation of p53. Although a number of previous studies have shown that HeLa cells are either p53 deficient (Ridgway et al., 1993) or with low expression level of p53 (Haupt et al., 1995), it has also been reported that in HeLa cells p53 could be functionally upregulated as evidenced by the increase of the p21 protein (Micheau et al., 2001). In fact, treatment using other indirubin derivatives have been observed to up-regulate p53 in human cancer cells (Lee et al., 2005; Ribas et al., 2006), implying a common mechanism in indirubin derivative-induced apoptosis. At present, it remains to be 75 further tested as how I3M induces p53 accumulation and activation, and whether the p53 activation observed is responsible for the up-regulation of DR4 and DR5. Another possible mechanism by which I3M promotes death receptor-mediated apoptosis is through modulation of NF-κB activity. The anti-apoptotic function of NFκB has been well established via the transcriptional regulation of various anti-apoptotic genes such as (c-FLIP, cIAP1/2, and Survivin ) (Micheau et al., 2001; Thome and Tschopp, 2001). Indirubin and its derivatives have been reported to inhibit the NF-κB signaling pathway stimulated by various activators, including TNFα, PMA and H2O2 (Sethi et al., 2006). In the study by Sethi and colleagues, I3M did not affect the basal level of NF-κB transcriptional activity. It remains to be further studied whether I3Mmediated caspase-8 activation is achieved via the suppression of the NF-κB signaling pathway. On the other hand, I3M-induced apoptosis in HeLa cells also exhibit a response typical of type II cells. While the extrinsic pathway is not sufficient for the full activation of effector caspases, the intrinsic mitochondrial pathway as demonstrated by caspase-9 activation (Fig. 3.4A) and cytochrome c release (Fig. 3.12) is involved; the permeabilization of mitochondria (or the loss of MMP) is the critical step in apoptosis that leads to an irreversible apoptotic stage. In the current study, evidence has shown that Bid cleavage (Fig. 3.8A) downstream of caspase-8 activation (Fig. 3.8B) has contributed partially to the apoptosis induced by I3M (Fig. 3.10B); Yet, there is still some doubt on how exactly the mitochondrial pathway is activated since blocking of the extrinsic pathway by synthetic caspase-8 inhibitor (Fig. 3.5), or even CrmA — a very specific caspase inhibitor (Fig. 3.13D), could not provide a complete protection, 76 which should be achieved if the mitochondria received apoptotic signal solely from the death receptor pathway. Therefore it is reasonable to believe some upstream apoptotic signal works directly on mitochondria besides caspase-8 activation. Other proapoptotic signals converge on mitochondria to induce outer mitochondrial membrane permeabilization include ROS stress or p53-dependent tumor suppression. Taking p53 as an example, on one hand, p53 can trigger apoptosis by inducing the expression of the BH3-only protein PUMA; on the other hand, p53 can act in a transcriptionindependent manner through direct activation of Bax or Bak or through binding to and blocking Bcl-2 and Bcl-XL (Green and Kroemer, 2004). Therefore, it would be interesting to know if the up-regulation of p53 induced by I3M has any direct effects on mitochondria in cancer cells. 4.3 Critical role of pro-apoptotic Bcl-2 family members BH3-only protein Bid plays critical role in I3M-induced apoptosis. Bid cleavage (Fig. 3.8A) acts downstream of caspase-8 activation (Fig. 3.8B) and mediates the transduction of apoptotic signal from the extrinsic pathway to the intrinsic pathway. Futhermore, Bax conformational change occurs as the consequences of caspase-8 activation (Fig. 3.11C) and Bid cleavage (Fig. 3.11D) based on immunofluorescence data using conformation-specific antibody 6A7. It is still not fully understood how the BH3-only proteins signal to the multi-domain members Bax and Bak, and together they increase the permeability of mitochondrial outer membrane (several hypotheses have been reviewed by Tsujimoto, 2003.). In addition to changes in Bid and Bax, the role of Bak is another crucial point worth studying although it was not investigated in our study; enhanced Bak protein level and oligomerization, as observed in 77 parthenolide-induced mitochondrial dysfunction (Zhang et al., 2004), can be a direction for a more detailed study. In addition to BH3-only proteins, the anti-apoptotic Bcl-2 family members are also known to modulate the pro-apoptotic activity of Bax through sequestrating Bax by the formation of heterodimers (Yi et al., 2003). In the present study, ectopic expression of Bcl-2 protein only offered moderate protection against I3M-induced cell death (Fig. 3.13C & D). Bcl-2 alone may not be sufficient to salvage the activation of the intrinsic pathway; collaboration of Bcl-2 and Bcl-XL would have a better protective effect since they have differential affinities for the pro-apoptotic members. Moreover, direct activation of caspase-3 by caspase-8 is not influenced by Bcl-2 overexpression. Hence the protective effect of Bcl-2 was not as significant as CrmA. Collectively these data suggest that the engagement of both the anti-apoptotic and pro-apoptotic Bcl-2 family members at the site of mitochondria is a key factor of the type II response in I3Minduced apoptosis of HeLa cells. 4.4 Sensitization of TRAIL-induced apoptosis The sensitizing effect of I3M on TRAIL-induced apoptosis in cancer cells enhances the therapeutic potential of I3M. Base on our data (Fig. 3.14), I3M can significantly increase and dramatically expedite the apoptosis induced by TRAIL in HeLa, CNE1 and HepG2 cells, but was not so effective in HT29 cells. TRAIL has the unique property of inducing apoptosis in cancer cells but sparing normal cells. The resistance to TRAIL found in many cancer cells limited its clinical application, which might involve aberrant expression of surface death receptors or changes in anti-apoptotic proteins (Fulda and Debatin, 2006). Therefore, anticancer agents that can sensitize 78 TRAIL-induced apoptosis work through either increased functionality of death receptors or inhibit the effectiveness of anti-apoptotic proteins. For example, Luteolin, a dietary flavonoid commonly found in some medicinal plants, sensitizes TRAILinduced apoptosis through down-regulation of XIAP (Shi et al., 2005). We have shown that I3M can increase the surface expression of DR4 and DR5 in HeLa cells (Fig. 3.7A), a process correlated to p53 up-regulation (Fig. 3.7C); therefore, the upregulation of death receptors induced by I3M reasonably contribute to the sensitizing effect on TRAIL-induced apoptosis, although the exact mechanism requires further study. Moreover, p53 is likely to play a critical role, since HT29, a p53 mutant cell line (Tao et al., 2007), was not significantly sensitized by I3M unless relatively high concentrations of I3M (20 µM) and TRAIL (5 ng/ml) were used (Fig. 3.14B). 4.5 Conclusions In summary, data from this study provide systematic evidence for apoptosis induced by I3M in three human cancer cell lines: cervical cancer HeLa, hepatoma HepG2 and colon cancer HCT116. More importantly, we reveal the apoptotic mechanism of I3M in HeLa cells: extrinsic death receptor pathway accompanied by type II response with critical involvement of the pro-apoptotic Bcl-2 family members (Bid and Bax). In addition, I3M is found to be effective in sensitizing TRAIL-induced apoptosis in various cancer cells. Indirubin and its derivatives have been known for their potential anti-tumor activities. Therefore understanding of such mechanisms provides the basis for future studies to expand the scope of their anti-cancer effects. 4.6 Directions for future study 79 Based on the observations of our study that I3M promotes the DR4 and DR5 expression and the sensitization effect of I3M on TRAIL-induced apoptosis in those TRAIL-resistant cancer cells, the major direction of future study would then be to answer the following questions: how I3M regulates p53, whether death receptor upregulation is the result of p53 activation, whether the expression level of anti-apoptotic proteins is influenced by I3M, and how these changes contribute to the enhanced apoptosis induced by TRAIL. 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Pharmacol Ther 92: 57-70. 91 [...]... mechanistic study, the demonstrated anti- angiogenic activity of I3M in human endothelial cells enhances the potential of I3M as an anti- cancer agent 14 Figure 1 .3 A schematic representation of the effect of I3M on TNF-induced NFκB activation and apoptosis (Sethi et al., 2006) 15 1.2.2 .3 In vivo Animal Model So far, only one rat tumor model has been established to assess indirubins’ antitumor activity in accessible... for 2 hr, the cellular content of indirubin and I3M (10 µM) did not differ significantly whereas indirubin- 5-sulfonate was not detectable in the cells even with high concentration (50 μM) Of all the cellular content, 93 ± 6% of indirubin was localized in the particulate fraction whereas 43 ± 14% of I3M was detected in the cytosol The highest growth inhibitory efficacy of I3M in the study above might be... that indirubin and I3M inhibit glycogen 2 A Qing Dai Indigo Blue B Figure 1.1 Qing Dai and chemical structures of indirubin derivatives A Photos of Indigo naturalis and indigo blue B Chemical structures of indigo, indirubin and indirubin derivatives (modified from Hoessel et al 1999 complimentary data) 3 synthase kinase -3 (GSK -3 ) (Leclerc et al., 2001; Meijer et al., 20 03) , and c-Src kinase (Stat3 signalling)... Hoessel et al., 1999 and Leclerc et al., 2001) Indirubin 5-chloroindirubin I3M Indirubin- 5sulphnic acid CDK1–cyclin B 10 0.4 0.18 0.055 CDK2–cyclin A 2.2 0.75 0.44 0. 035 CDK2–cyclin E 7.5 0.55 0.250 0.15 CDK4–cyclin D1 12 6.5 3. 33 0 .3 CDK5–p35 5.5 0.8 0.1 0.065 Erk1 > 100 > 100 > 100 38 Erk2 43 > 100 > 100 > 100 c-Raf > 10 > 10 > 100 5.5 MAPKK > 100 > 100 > 100 3 c-Jun N-terminal Kinase > 100 21 N.A 5.2... study carried out in rat tumor model provides further evidence for indirubins’ anti- tumor activity (Kim et al., 2007) In the attempt to reveal the mechanism of action of indirubins, various biological activities of indirubin and its derivatives have been discovered It has been well established that indirubin and indirubin- 3 -monoxime (I3M) are strong inhibitors for cyclin-dependent kinases (CDKs) (Hoessel... the anti- tumor activity of indirubins (Kim et al., 2007) 1.2.2.1 Clinical trials Indirubin was approved for clinical trials against chronic myelocytic leukaemia (CML) and chronic granulocytic leukaemia (Institute of Haematology, 1979) In one of these clinical trials, 26% of the 31 4 CML patients showed complete remission under indirubin treatment and 33 % showed partial remission (Ma and Yao, 19 83) Pharmacokinetics... which is also NADPH-dependent 1.2 Pharmacological mechanisms of Indirubin and it derivatives 1.2.1 Anti- inflammatory activity 5 The anti- inflammatory activity of indirubin is mainly reflected by its anti- virus activity Indirubin can inhibit viral replication or decrease viral yield For example, I3M has been reported to inhibit Tat-induced replication of HIV-1 (primary and drug-resistance strains) RNA... inhibited by I3M include proteins involved in anti- apoptosis, proliferation (cyclin D1 and c-Myc), and invasion (Fig 1 .3) I3M can also suppress cytokine-induced cellular invasion The understanding of I3M as a potent inhibitor of NFκB fortifies its promises as a therapeutic agent 1.2.2.2.4 Anti- angiogenesis A recent study reported that I3M had anti- angiogenic activity based on an automated, quantitative... signalling pathway Stat3 signalling pathway has an important role in oncogenesis and therefore Stat3 protein is a promising anticancer target Three indirubin derivatives have been shown to potently block constitutive Stat3 signalling in human breast cancer cells (MDA-MB468/- 435 ) and prostate cancer cells (DU145) Mechanistic study revealed that the inhibition of Src kinase by indirubin derivatives led... decreased tyrosyl phosphorylation of Stat3 and thereby suppressed Stat3 DNA binding-activity, which ultimately affected the target genes of Stat3, such as the anti- apoptotic protein Mcl-1 and Survivin (Nam et al., 2005) 1.2 .3. 3 Aryl hydrocarbon receptor (AhR) 18 Aryl hydrocarbon receptor (AhR), a member of the bHLH/PAS family of transcriptional factors, mediates the responses of some xenobiotics including