Inhibition of misfolded n cor induced survival pathway in APL by artemisinin

INHIBITION OF MISFOLDED N-COR INDUCED SURVIVAL PATHWAY IN APL BY ARTEMISININ YEO HUI LING ANGIE NATIONAL UNIVERSITY OF SINGAPORE 2011 INHIBITION OF MISFOLDED N-COR INDUCED SURVIVAL PATHWAY IN APL BY ARTEMISININ YEO HUI LING ANGIE (B.Sc.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MEDICINE YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2011 ! I ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Dr Matiullah Khan, for his patient guidance, advice and support throughout this course of work. I am grateful for the opportunity to work and learn here. My gratitude extends to Cancer Science Institute, where this project was carried out. My sincere thanks go to Dr Azhar Ali and Dr Angela Ng for their insightful discussion and technical advice. The knowledge that they shared from their scientific expertise and life experiences have been very motivating and enriching. I would like to thank all present and ex-members of this lab: Angela, Azhar, Dawn, Jess, Li Feng, Lizan, Su Yin and Wan Qiu. Thank you for your company and support. It has been a pleasure working with everyone. I would like to thank my other friends in the laboratory: Meg, Li Ren, Pei Li, Seow Ching, Ben, John, Sarawut and Seetha, for their friendship and encouragement. Meg, thank you for your friendship and for being there in every way possible. Li Ren, thank you for your help and company during those late hours and weekends. Su Yin, thank you for being on this journey with me. I would also like to thank Rikki for teaching me how to use the FACS machine. I am grateful to have such wonderful friends and colleagues. Your friendship and encouragement have pulled me through whenever I was down. My sincere appreciation goes to my current boss, Dr Ho Han Kiat. His kind understanding and advice has enabled me to finish this thesis. I would also like to express my heartfelt thanks to Dr Ho and Dr Azhar Ali for taking their precious time off to proof-read this thesis. Lastly, I would like to thank my family for their unconditional love and support that has encouraged me never to give up. My heartfelt thanks go to my parents who are always there for me. ! II Thank you to everyone who has contributed to the success of this thesis in one way or another Yeo Hui Ling, Angie November 2011 III! ! TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS III SUMMARY VI LIST OF TABLES VII LIST OF FIGURES VIII ABBREVIATIONS X Chapter 1: Introduction 1 1.1 Leukemia 1 1.2 Acute promyelocytic leukemia (APL) 2 1.2.1 Role of PML-RARα in APL 3 1.2.2 N-CoR and its role in APL 5 1.2.3 Current knowledge of the role of PML-RARα and N-CoR in the pathogenesis of APL 8 1.2.4 Current treatment strategies for APL 9 1.2.5 Rationale for the need of new therapeutic options 10 1.2.6 Artemisinin: a candidate drug for APL 11 1.3 1.4 ! ER stress and protein folding 12 1.3.1 Protein folding 12 1.3.2 The ubiquitin-proteasome proteolytic pathway 14 Autophagy 15 1.4.1 Macroautophagy 15 1.4.2 Chaperone-mediated autophagy 18 ! IV! ! 1.5 PI3K/Akt survival pathway 19 1.6 Project hypothesis and objectives 20 1.6.1 Current perspective in APL 20 1.6.2 Hypothesis and objectives 23 Chapter 2: Materials and Methods 25 2.1 Materials 25 2.1.1 Cell lines 25 2.1.2 Drugs 25 2.1.3 Antibodies 26 2.1.4 Primers 28 2.2 ! Methods 28 2.2.1 Cell culture 28 2.2.2 Transfection 28 2.2.3 Cell proliferation assay 29 2.2.4 Cell lysis for protein extraction 29 2.2.5 Western blotting 30 2.2.6 Polyacrylamide gels 31 2.2.7 Coomassie staining 31 2.2.8 RNA extraction 32 2.2.9 Reverse-transcription polymerase chain reaction (RT-PCR) 32 2.2.10 Flow cytometry apoptosis assay 33 2.2.11 Proteasome sensor assay 34 2.2.12 Immunostaining and fluorescence microscopy 34 2.2.13 Measurement of internal ATP levels 35 ! V! ! Chapter 3: Results 36 3.1 Artemisinin selectively inhibits the growth of APL but not non-APL cells 36 3.2 Artemisinin derivative, GC011, promotes apoptosis of APL cells 42 3.3 Artemisinin derivative, GC011, promotes the degradation of misfolded N-CoR and PML-RARα 44 3.4 Degradation of N-CoR by GC011 via the proteasome-dependent pathway in APL cells 49 3.4.1 GC011-induced N-CoR degradation is mediated via the proteasome pathway 49 3.4.2 N-CoR is rescued by MG132 in GC011-treated APL cells 52 3.5 3.6 Autophagy is blocked by GC011 in APL cells 54 3.5.1 Autophagy is activated in APL cells and contributes to cellular growth 54 3.5.2 GC011 blocks autophagy in NB4 cells 59 3.5.3 GC011-induced degradation of N-CoR is associated with a decrease in intracellular energy 60 GC011 blocks autophagy via the PI3K/Akt survival pathway in APL cells 62 Chapter 4: Discussion 64 4.1 Artemisinin shows promise as a therapeutic agent in APL 64 4.2 GC011 induces caspase-activated apoptotic pathways in APL 65 4.3 GC011 enhances N-CoR degradation through the proteasome pathway 66 4.4 GC011 inhibits autophagy in NB4 cells 68 4.5 GC011 inhibits the PI3K/Akt pathway in NB4 cells 69 4.6 Hypothesis model for the action of GC011 70 REFERENCES ! 74 ! VI! ! SUMMARY Acute promyelocytic leukemia (APL) is characterized by PML-RARα, a fusion protein resulting from a chromosomal translocation between the promyelocytic leukemia (PML) gene and retinoic acid receptor α (RARα) gene. PML-RARα was shown to promote misfolding and accumulation of nuclear receptor co-repressor (N-CoR) in the endoplasmic reticulum (ER) and cause unfolded protein response (UPR)-linked apoptosis. However in APL cells, N-CoR was found to be degraded, relieving ER stress and escaping cell death. Previous results also showed that autophagy was elevated in APL cells and drug inhibition of autophagy led to a stabilization of N-CoR with corresponding decrease in adenosine triphosphate (ATP) levels, suggesting a possible function of N-CoR where APL cells may use its degradation through autophagy to provide an alternative energy source for cancer cell survival. Here, I report a drug artemisinin as a potential therapeutic agent which selectively promotes growth inhibition and apoptosis in APL cells. Artemisinin enhanced the degradation of N-CoR, which could be restabilized by treatment with a proteasome inhibitor. Levels of autophagic and survival markers, and ATP in APL cells also decreased after artemisinin treatment. These findings suggest that artemisinin possibly enhances the proteasomal degradation of misfolded N-CoR, thus depriving cancer cells of the extra energy source generated by the autophagic degradation of misfolded proteins. !VII! ! LIST OF TABLES Table 1.1 Summary of transcription factors interacting with N-CoR and their roles in cellular processes Table 2.1 Steps for PCR amplification 7 33 VIII! ! ! LIST OF FIGURES Figure 1.1 Suggested model of PML-RARα action in APL 5 Figure 1.2 The domains of N-CoR 6 Figure 1.3 Molecular circuitry and signaling pathways regulating autophagy 18 Figure 1.4 Representation of the regulation of ER stress and UPR in APL cells 22 Figure 1.5 Proposed mechanism of the effects of misfolded N-CoR in APL and non-APL cells 24 Figure 2.1 Chemical structures of synthesized artemisinin derivatives 26 Figure 3.1 Artemisinin derivatives inhibit proliferation of NB4 cells Figure 3.2 GC011 inhibits cell proliferation of RA-sensitive and RAresistant APL cell lines 40 Figure 3.3 Artemisinin derivatives selectively inhibit proliferation of APL cells 41 Figure 3.4 GC011 induces apoptosis in NB4 cells 42 Figure 3.5 GC011 activates the apoptotic pathway in NB4 cells 43 Figure 3.6 GC011 enhanced the degradation of N-CoR and PML-RARα in NB4 cells 45 Figure 3.7 GC011 induced the degradation of transfected N-CoR and PML-RARα in 293T cells in a dose-dependent manner 46 Figure 3.8 GC011 reduced the expression of transfected N-CoR and PML-RARα in the cytosol of 293T cells in a dose-dependent manner Figure 3.9 GC011 induced a significant down-regulation of N-CoR in APL cells but not non-APL cells 37-39 47-48 49 ! IX! ! Figure 3.10 GC011 did not cause significant change to mRNA levels of NCoR in NB4 cells 50 Figure 3.11 GC011 enhanced the degradation of the proteasome sensor in 293T cells in a dose-dependent manner 51 Figure 3.12 MG132 reversed GC011-induced N-CoR degradation in NB4 cells 53 Figure 3.13 MG132 reversed GC011-induced N-CoR degradation in 293T cells 54 Figure 3.14 LC3-II/LC3-I ratio is high in APL cells 56 Figure 3.15 BA-1 reduces the intracellular ATP level in NB4 cells in a dose-dependent manner 57 Figure 3.16 APL cells are resistant to glucose starvation-induced growth inhibition 58 Figure 3.17 GC011 inhibits autophagy in NB4 cells 59 Figure 3.18 Reduction of intracellular ATP levels is associated with GC011-induced N-CoR degradation in NB4 cells 61 Figure 3.19 GC011 inhibits the PI3K/Akt pathway in NB4 cells 63 Figure 4.1 Schematic model of hypothesis in APL cells 72 ! X ! ABBREVIATIONS ALL acute lymphoid leukemia AML acute myeloid leukemia APL acute promyelocytic leukemia Atg autophagy-related gene ATO arsenic trioxide ATRA all-trans retinoic acid BA-1 Bafilomycin A1 BSA bovine serum albumin Ca2+ calcium CML chronic myelogenous leukemia CO2 carbon dioxide CR complete remission DAPI 4, 6-diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DS differentiation syndrome ER endoplasmic reticulum FBS fetal bovine serum GFP green fluorescent protein HDAC histone deacetylase HRP horseradish peroxidase Hsp heat shock protein XI ! hr hours kDa kilo Dalton min minutes mRNA messenger RNA mTOR rapamycin MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N-CoR nuclear receptor co-repressor OSGEP O-sialoglycoprotein endopeptidase PBS phosphate buffered saline PI3K phosphotidyl inositol 3-kinase PML promyelocytic leukemia POD PML oncogenic domain PVDF polyvinylidene difluoride RA retinoic acid RARα retinoic acid receptor α RAREs retinoic acid response elements RPMI Roswell Park Memorial Institute RT-PCR reverse transcription polymerase chain reaction s second SDS sodium dodecyl sulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis SMRT silencing mediator of retinoic and thyroid receptors UPR unfolded protein response WHO World Health Organization 1 ! Chapter 1: Introduction 1.1 Leukemia Leukemia, a hematological malignancy, is caused by an abnormal increase in leukocytes produced in the bone marrow. It can be categorized as acute or chronic, depending on the maturity of cancer cells which affects the progression of disease. Acute leukemia is characterized by the rapid expansion of undifferentiated precursor cells, while chronic leukemia is characterized by excessive accumulation of mature white blood cells. Leukemia is subdivided into lymphoid or myeloid leukemia, depending on the lineage of hematopoietic cells affected. Lymphocytic leukemia mainly consists of lymphocytes like early B-cells, precursor B-cells and precursor Tcells. Myeloid leukemia involves myeloid cells like early myeloblasts, myeloblasts, promyelocytes, myelocytes and neutrophils, monoblasts and monocytes, megakaryoblasts, or erythroid precursors. Collectively, there are four main types of leukemia – acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). ALL is the most common leukemia in children while AML and CLL are most common in adults [1]. There are many subtypes of AML and the French-American-British (FAB) Cooperation Group’s classification system based on morphological features is widely used [2]. A newer and improved classification by the World Health Organization (WHO) is also being used. This classification incorporates cytogenetic results that links the FAB subtypes to associated chromosomal translocations [3]. Reciprocal translocations between non-homologous chromosomes have been implicated in various diseases including leukemia [4]. A common chromosomal abnormality is the t(9;22) translocation between the Abl1 and BCR genes, which is the 2 ! hallmark of CML and ALL. Chromosomal translocations can be grouped into seven subtypes – translocations involving the MLL gene (AML), CBF and TEL/ETV6 genes (childhood ALL, AML, CML), retinoic acid receptor α (RARα) (AML), E2A gene (ALL), tyrosine kinases (CML), nucleoporins (AML) and immunoglobins or T-cell receptors (ALL). A comprehensive list of recurring chromosome translocations in leukemia can be found in Table 1 in [5]. 1.2 Acute promyelocytic leukemia (APL) APL is classified as AML-M3 under the FAB classification system [2]. It accounts for about 10-15% of all AML cases in adults [6], with a lower incidence in children [7]. APL is characterized by the fusion protein PML-RARα, which is a result of a reciprocal translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and retinoic acid receptor (RAR) gene on chromosome 17. PMLRARα can be found in 98% of APL cases [8, 9]. Other rare forms of APL involve the fusions of RARα to promyelocytic leukemia zinc finger (PLZF), nucleophosmin (NPM), nuclear matrix-associated (NuMA) and signal transducer and activator of transcription 5b (STAT5b) [10]. APL associated with these fusion proteins are responsive to all-trans retinoic acid (ATRA), with the exception of PLZF-RARα and possibly STAT5b-RARα- associated APL, which are ATRA-resistant [11, 12]. Morphologically, APL is characterized by the arrest of leukemic cells at the promyelocytic stage during granulocytic differentiation [13]. According to the FAB, there are two main cytological subtypes, the classical hypergranular form and the variant microgranular form [2, 14]. The hypergranular form features numerous coarse granules in the cytoplasm, where multiple Auer bodies are also commonly found. 3 ! Leukemic cells of the microgranular form have sparse granules with bilobed nuclei [13]. APL is also characterized by the disintegration of PML oncogenic domains (PODs) or nuclear bodies (NBs), which are dot-like structures in the nucleus [15, 16]. PODs have been proposed to act as organizing centers for the regulation of several important cellular processes like transcription and development [17-19]. Thus the disintegration of PODs is hypothesized to be linked to the differentiation arrest of APL cells [20]. 1.2.1 Role of PML-RARα in APL As mentioned earlier, PML-RARα fuses the N-terminus multimerization domain of PML to the DNA and ligand-binding domains of RARα [10, 21]. While the RARα portion remains constant, different breakpoint cluster regions (bcr1-3) in PML give rise to fusion proteins of different lengths. Majority of the APL patients exhibit the long PML-RARα resulting from breakage at bcr1. Breakage at bcr2 yields an intermediate length of PML and bcr3 yields the shortest PML [10]. Detection of these PML-RARα transcripts allows for a sensitive and specific test for the diagnosis of APL [22]. There have been multiple studies on the role of PML-RARα in the pathogenesis of APL and PML-RARα has been proposed to act as a dominant negative transcription repressor of RA target genes essential for promyelocyte differentiation [20]. PML-RARα disrupts the normal functions of both PML and RARα. It can form homodimers and also heterodimerizes with PML and RXR separately. Unlike wildtype RARα, which requires binding to RXR to bind DNA, PML-RARα homodimers can bind to retinoic acid response elements (RAREs) and 4 ! act as constitutive repressors [10, 23]. Nuclear corepressor complexes containing NCoR [24] and silencing mediator of retinoic and thyroid receptors (SMRT) [25], and histone methyltransferases [26] are recruited to the promoters of RA target genes, resulting in transcriptional repression of genes required for differentiation of granulocytes. Pharmacological concentrations of ATRA is needed to dissociate NCoR from PML-RARα, an event that destabilizes and promotes the degradation of the latter protein [27]. While normal PML is localized to NBs, it is found to be delocalized to microspeckles after forming heterodimers with PML-RARα [15, 16]. PML-RARα may also draw other nuclear proteins like RXR and Rb into the microspeckles [16, 28]. In addition, Daniel et al. found that a large proportion of PML-RARα was localized in the cytoplasm instead of the nucleus, strengthening the idea that PMLRARα could draw critical factors from RAR target genes [10, 29]. There is also a gain-of-function of PML-RARα. Through the PML moiety, PML-RARα is able to bind to a large variety of de novo target sequences that were previously not efficiently recognized by the normal RXR-RARα heterodimers. This leads to the transcriptional deregulation of sites recognized by other nuclear receptors controlling processes such as myeloid differentiation or stem-cell renewal [23, 30]. 5 ! Figure 1.1. Suggested model of PML-RARα action in APL [10]. 1.2.2 N-CoR and its role in APL Nuclear receptor co-repressor (N-CoR) is a 270 kDa protein that was discovered with SMRT as interacting partners and mediators of the repressive functions of unliganded RAR and thyroid hormone receptor (TR) [31, 32]. Both proteins contain nuclear receptor interaction domains (NRIDs), multiple repressor domains (RDs) and Swi3/Ada2/N-CoR/TFIIIB (SANT) motifs [31-35]. SANT motifs are postulated to act as histone binding modules and RDs may serve as binding platforms for the various enzymes like histone deactylases (HDACs) recruited to repress gene promoters. Although N-CoR and SMRT are similar, their functions are not redundant as N-CoR deficient mice have been shown to be embryonic lethal [33]. 6 ! Figure 1.2. The domains of N-CoR. Repression domains (RI, RII, RIII) and SANT domains (A and B) are indicated, as are interaction domains for HDACs, nuclear receptors (I and II) and other transcription factors [36]. N-CoR and SMRT can form complexes with many proteins. These proteins that were consistently found in a complex with N-CoR/SMRT include HDAC3, transducin β-like 1 (TBL1), the TBL1-related protein (TBLR1) and G protein pathway suppressor 2 (GPS-2) [37-40]. TBL1, TBLR1 and GPS-2 help to regulate the stability and activity of the corepressor complex. TBL1 and TBLR1 mediate the proteasome-dependent degradation of N-CoR/SMRT complexes from promoters, to allow de-repression of the gene and recruitment of coactivators [41]. One major function of N-CoR and SMRT is to repress gene transcription. N-CoR and SMRT binds and activates HDAC3 through their deacetylase activating domain (DAD) [42]. HDAC3 then mediates the deactylation of lysines on the histone tails of target promoters to promote repression [43]. A ‘feed-forward mechanism’ of repression by the N-CoR/SMRT complex has been proposed by Yoon et al [40]. Current models suggest that the corepressor complex binds acetylated chromatin and deacetylates the histone tails. The complex shows an increased binding affinity for the hypoacetylated product, thus enhancing gene repression [43]. The N-CoR/SMRT complex can also 7 ! interact with Ski, Sno and mSin3 to regulate the tumour suppressor Mad-mediated transcriptional repression [44]. Together, the N-CoR/SMRT complex and HDAC3 facilitate transcriptional repression by various transcription factors to regulate multiple cellular processes like differentiation, proliferation and apoptosis [36]. They also play a role in development, metabolism and inflammation [45]. A list of the interacting transcriptional factors and their roles is shown in Table 1.1 [36]. Table 1.1 Summary of transcription factors interacting with N-CoR and their roles in cellular processes Transcription factor Role in cellular processes POU homeodomain factors Development, differentiation of pituitary cells Pit1 Homeobox factor PBX Determiner of cell fate and segment identity Bcl-6 Apoptosis MAD, MyoD and HES- Suppress proliferation, induce terminal differentiation related repressor proteins (HERPs) Su(H)/RBP-J/CBF1 Differentiation, proliferation, apoptosis N-CoR has been implicated in cancers and neuronal diseases. In various leukemias like APL and AML, PML-RARα and AML1-ETO fusion proteins bind to the N-CoR/SMRT histone deacetylase complex, resulting in gene repression that blocks differentiation and allow uncontrolled growth of hematopoietic cells [45]. In Huntington’s disease, N-CoR is localized with mSin3 exclusively in the cytoplasm of the cortex and caudate, while in the normal brain, both proteins are localized in both nucleus and cytoplasm. This suggests that relocalization of N-CoR results in alteration of transcription and pathogenesis of disease [46]. Recently, N-CoR has also found to be involved in glioblastoma multiforme (GBM). Further, increased nuclear N-CoR 8 ! expression has been found in severe grades of astrocytomas, where it maintains tumour cells in an undifferentiated state [47]. 1.2.3 Current knowledge of the role of PML-RARα and N-CoR in APL pathogenesis As mentioned earlier, N-CoR is involved in the regulation of multiple biological processes and is essential for Mad-mediated transcriptional repression, which is responsible for the regulation of the growth and maturation of myeloid cells. PML-RARα inhibits this Mad- and Rb- mediated transcriptional repression and leads to transformation of APL. Deletion of two N-CoR interacting sites in PML-RARα, the coiled-coil domain on PML and CoR-box on RAR, prevents this inhibition [48, 49], suggesting that PML-RARα may bind aberrantly to N-CoR and lead to a loss of function. Natively folded N-CoR normally localizes in the nucleus when associated with PML or RAR protein and is also detergent-soluble [50]. However, significant levels of PML-RARα are found in the cytoplasm [15] and it is hypothesized that the cytoplasmic PML-RARα may bind N-CoR and promote its conformational change, causing it to accumulate as insoluble protein aggregates in the endoplasmic reticulum (ER) [50]. Besides leading to a loss of N-CoR function by lifting the repression of selfrenewal genes, there may also be a gain of function of N-CoR. It has been observed in APL cells that there are two distinct forms of PML-RARα and N-CoR, which is nuclear and cytosolic. Similar to cytosolic PML-RARα engaging the cytosolic form of N-CoR, it is likely that nuclear PML-RARα recruits the native N-CoR to turn on the expression of RA target genes [20]. The combined deregulation of the two pathways eventually contributes to transformation of APL. 9 ! 1.2.4 Current treatment strategies for APL APL was first treated in the 1970s with anthracyclins or anthracyclins combined with cytarabine (Ara-C). Previous reports have shown that daunorubicin and idarubicin used as single agents induced complete remission (CR) in 55-88% of patients [51, 52]. The introduction of ATRA, a non-cytotoxic differentiating agent, by a Shanghai group in 1998, has led to an improved prognosis of APL with a better long-term outcome [53]. CR of up to 90% was observed and the biologic signs of coagulopathy also improved. CR was achieved through the differentiation of APL blasts to mature granulocytes [54-56]. However, most patients were found to relapse with just ATRA treatment alone [54]. Currently, the standard induction therapy for APL is based on the combination of ATRA and chemotherapy [57, 58]. Combining both therapies has been reported to reduce the incidence of relapse and allows for a more effective control of ATRAinduced leukocytosis, thus reducing the incidence and severity of ATRA syndrome [54, 57]. ATRA syndrome is a potentially fatal occurrence which can result from treatment with ATRA. ATRA degrades PML-RARα, which contributes to remission of APL and degradation of PML-RARα occurs via three pathways. First, proteases activated by RA-induced differentiation cleave the PML moiety of PML-RARα [27, 59]. Second, RA-induced transcriptional activation is coupled to proteasome-mediated RARα degradation [60] while the third pathway involves degradation through the mTOR autophagic pathway [61]. Arsenic trioxide (ATO) is an effective therapy for relapsed patients who were treated with the ATRA/chemotherapy combination therapy. Studies have shown ATO to induce CR in 80-90% of relapsed patients [62]. Shen et al. also reported that combination therapy of ATRA and ATO was effective in achieving a similar CR rate 10 ! within a shorter period of time [63]. ATO has a dual mechanism of action where it induces differentiation of APL cells at low concentrations and apoptosis at high concentrations [57]. Like ATRA, ATO also degrades PML-RARα but via degradation of the PML moiety, along with normal PML. It targets PML-RARα and PML into nuclear bodies before inducing degradation. There are two mechanisms by which nuclear body formation takes place. First, ATO induces the formation of reactive oxygen species (ROS) [64], which causes multimerization of PML, targeting to nuclear bodies and PML sumoylation by ubiquitin-conjugating enzyme 9 (UBC9) [65]. Second, ATO can also bind PML cysteines directly [65, 66], enhancing UBC9 binding to the PML RING finger and ultimately PML sumoylation [66]. PML sumoylation results in the recruitment of the SUMO-dependent ubiquitin ligase and RING finger protein 4 (RNF4) to PML nuclear bodies. RNF4 poly-ubiquitylates PML and targets it to the proteasome for degradation [67, 68]. Degradation of PML-RARα by ATRA and ATO relieves the transcriptional repression by the fusion protein and allows for normal regulation of RARα-responsive genes to induce myeloid differentiation [69]. 1.2.5 Rationale for the need of new therapeutic options Although current treatments for APL including ATRA, ATO and chemotherapy have proven to be very effective and are able to induce high CR rates, there are a few drawbacks which warrant the need to develop new therapeutic agents. One factor is the relapse of patients after CR. Relapse occurs in 5-30% of APL patients treated with ATRA and chemotherapy. The relapse rate is higher in high-risk patients with high white blood cell (WBC) count [52, 62]. 11 ! Another factor to be considered is the side effects of ATRA and ATO treatment. ATRA can lead to major blood hyperleukocytosis [70] and the potentially fatal differentiation syndrome (DS) (formerly known as ATRA syndrome) [71]. Symptoms include dyspnea, unexplained fever, weight gain, peripheral edema, unexplained hypotension, acute renal failure or congestive heart failure, and particularly if a chest radiograph demonstrates interstitial pulmonary infiltrates or pleuropericardial effusion [72]. Occurrence of DS is also associated with an increased risk of subsequent relapse. Currently, no significant prognostic markers have been found for the prediction for DS [73].In addition to DS, ATO is also associated with cardiac arrhythmia [74] and electrolyte abnormalities [54]. Secondary resistance occurs in all patients treated with ATRA [75]. Hence ATRA needs to be used in combination with chemotherapy, and this may subject the patients to cardiac toxicity in the long-term [54, 76]. Thus, it is crucial to develop new therapeutic agents that specifically target APL cells, reduce relapse rates, and also to reduce chemotherapy-associated toxicity. 1.2.6 Artemisinin: a candidate drug for APL Artemisinin is a sesquiterpene lactone isolated from the Artemisia annua plant. It has been used in Chinese traditional medicine for 2000 years in the treatment of fever and malaria [77]. Besides isolation from the plant, artemisinin has also been produced from Saccharomyces cerevisiae engineered to produce the artemisinin precursor, artemisinic acid [78]. Artemisinin and its derivatives are currently recommended by the World Health Organization (WHO) for the treatment of Plasmodium falciparum strains of malaria which have developed resistance to traditional anti-malarial drugs like 12 ! chloroquine and sulfadoxine-pyrimethamine [79, 80]. Due to the short half-life of the drug, artemisinin derivatives are commonly used in combination with another longeracting drug, known as artemisinin-based combination therapy (ACT). Current ACTs use the artemisinin derivatives such as artemether, artesunate or dihydroartemisinin. These are chemically modified analogues synthesised to improve the bioavailability of artemisinin [81]. In addition to being an effective anti-malarial drug, artemisinin and its derivatives have also been found to exhibit anti-cancer properties like arresting the growth or inducing apoptosis of cancer cells. The Developmental Therapeutics Program of the National Cancer Institute in USA analysed 55 human cancer cell lines and showed that artesunate has strong anti-cancer activity against many cancer cell lines like leukemia, colon cancer, melanomas, breast, ovarian, prostate, central nervous system and renal cancer cell lines [82]. Another artemisinin derivative, dihydroartemisinin, has also been shown to inhibit the growth of human ovarian cancer cells and sensitise them to carboplatin therapy [83]. There are various mechanisms by which artemisinin exert its anti-proliferative effect. It may induce apoptosis by activating caspase 3, increasing poly ADP-ribose polymerase (PARP) and the Bax/Bcl-2 ratio, and downregulating Mdm2. It can also downregulate the transcription of Cdk4 to block cell cycle progression [84]. 1.3 ER stress and protein folding 1.3.1 Protein folding Protein folding is a process essential for cellular function. Secreted, membrane-bound and organelle-targeted proteins are synthesized and folded in the endoplasmic reticulum (ER) [85]. Newly synthesized polypeptide chains are 13 ! translocated into the ER and begin folding as they are co-translationally modified with the addition of disulphide bonds or N-linked glycans [86, 87]. The ER has an oxidizing environment containing high amounts of adenosine triphosphate (ATP) and Ca2+ for its proper function [85]. Chaperones are key components that regulate folding of proteins. They assist in the folding of newly translated proteins, refolding of misfolded proteins, prevent aggregation and facilitate proteolytic degradation [88]. In the ER, three classes of proteins mediate the folding of nascent proteins – foldases, molecular chaperones and lectins [89]. Foldases like protein disulphide isomerase (PDI) and peptidyl prolyl cis-trans-isomerase (PPI) accelerate the ratelimiting steps of the folding process by catalyzing the formation of disulphide bonds or isomerization of peptide bonds proximal to proline residues respectively [90]. Common molecular chaperones include GRP78 (also known as BiP) and GRP94. GRP78 belongs to the heat shock protein (Hsp) 70 chaperone family. It consists of an N-terminal ATPase and a C-terminal substrate binding domain. Conformational changes in GRP78 regulate its binding affinity for peptides in an ATP-dependent manner. The ATP-bound state allows for peptide binding, which is stabilized when ATP is hydrolyzed to ADP. PDI then promotes disulphide reduction and rearrangement until proper folding is achieved. Lastly, nucleotide exchange factors catalyze the ADP-ATP exchange and the folded protein is released [88, 91]. Partially folded monoglucosylated N-linked glycans are recognized by the lectins, calnexin (CNX) and its soluble homologue calreticulin (CRT) [92]. CNX and CRT bind to thiol oxidoreductase ERp57 [93] to facilitate disulphide bond formation [94]. Glucosidase II removes the glucose residue and enables the release of glycoproteins from the lectins. If folding is not completed, the glycoprotein is reglucosylated by UDP-glucose glycoprotein glucosyltransferase (UGGT) and the 14 ! cycle of binding and release from the lectins can repeat until proper folding is achieved [95, 96]. Improperly folded proteins may ultimately be targeted for proteasomal degradation by ER degradation-enhancing α-mannosidase-like protein (EMEM), which removes the mannose residue [97, 98]. ER is the place for de novo folding of proteins and also refolding of misfolded proteins. Otherwise, misfolded proteins are translocated in a retrograde fashion to the cytosol for refolding by cytosolic Hsp family chaperones or targeted for degradation by the 26S proteasome [99]. 1.3.2 The ubiquitin-proteasome proteolytic pathway The ubiquitin-proteasome system (UPS) is a major regulatory pathway involved in protein degradation. It is responsible for the controlled degradation of proteins involved in cellular processes like cell cycle, DNA repair, immune and inflammation response as well as response to stress to maintain normal cellular homeostasis [100]. Hence, deregulation of the proteosomal pathway results in diseases like neurodegenerative diseases and cancers [101]. In the ER, misfolded proteins are usually refolded by chaperones. When refolding fails, the misfolded proteins are usually degraded by ER proteinasesand either directed to the lysosome or transported back to the cytosol for proteosomal degradation [102]. Proteins targeted for recognition and degradation by the 26S proteasome undergo covalent attachment of multiple ubiquitin molecules. This ubiquitination process proceeds in a three-step mechanism. The first step involves the ATP-dependent activation of the C-terminal glycine of ubiquitin by E1 (ubiquitinactivating enzyme) to a thiol ester intermediate. Next, E2 (ubiquitin-conjugating enzyme) transfers the ubiquitin molecule from E1 to the ubiquitin-protein ligase, E3- 15 ! bound protein substrate. Lastly, the ubiquitin-tagged proteins are degraded by proteases in the 26S proteasome [103]. 1.4 Autophagy Autophagy is a catabolic degradation process important for homeostatic functions like protein degradation and organelle turnover. It generally plays a housekeeping role by degrading misfolded or aggregated proteins, damaged organelles and eliminating intracellular pathogens. It is also upregulated under conditions of cellular stress like nutrient deprivation, providing an alternative source of intracellular building blocks and maintaining energy production for cell survival [104]. In cancer, this may contribute to tumor growth and therapeutic resistance [105]. There are three main types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Microautophagy involves the direct uptake of cytosolic components by the lysosome itself through invagination of the lysosome membrane [106]. However, the role of microautophagy in higher eukaryotes remains unclear. Macroautophagy and chaperone-mediated autophagy will be discussed in the following sections. 1.4.1 Macroautophagy Macroautophagy (hereafter referred to as autophagy) is characterized by the formation of double-membrane vesicles, known as autophagosomes, that engulfs cytoplasmic cargo. Autophagosomes then fuse with lysosomes, forming autolysosomes, where the cargo is then degraded by lysosomal enzymes. After degradation, lysosomal proteases and transporters export amino acids and other byproducts back out to the cytoplasm to be re-used for building macromolecules or for 16 ! metabolism [107]. Thus autophagy promotes energy efficiency through ATP generation and prevents cellular damage by removing non-functional proteins and organelles [106]. Autophagy has been widely studied in the yeast system and is regulated by many proteins like the autophagy-related genes (Atg) proteins. Many mammalian homologs of the Atg proteins have been found. Phagophore formation in mammalian systems is regulated by vesicular protein sorting 34 (Vps34), a class III phosphatidyl inositol 3-kinase (PI3K), and its binding partner Atg6/Beclin-1. The complex is essential for phagophore elongation and recruitment of other Atg proteins [108]. Other regulatory proteins also complex with Vps34 and Beclin-1 to either promote autophagy, such as UVRAG, BIF-1, Atg14L and Ambra [109, 110], or inhibit autophagy, such as Rubicon and Bcl-2 [111, 112]. The interaction of Bcl-2 and Beclin-1 on the regulation of autophagy has been well characterized. Beclin-1 interacts with Bcl-2 via the BH3 domain, which disrupts its binding with Vsp34 and results in the inhibition of autophagy [112]. When cells undergo starvation, JNK1mediated phosphorylation of Bcl-2 releases Beclin-1 to interact with Vsp34 to promote autophagy [113]. In contrast to class III PI3K, class I PI3K has an inhibitory effect on autophagy. Class I PI3K generates phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) and activates Akt, which in turn activates mammalian target of rapamycin (mTOR) [114]. mTOR downregulates autophagy by phosphorylating Atg13, preventing it from interacting with Ulk1 and inhibiting the formation of a trimeric complex required for autophagosome formation [115]. mTOR is a main regulator of autophagy. Under conditions of nutrient deprivation, hypoxia and low ATP levels, adenosine monophosphate kinase (AMPK) is activated, which represses 17 ! mTOR to initiate autophagy [116]. mTOR is also inhibited when there is reduced Akt activity in response to reduced growth factor receptor activity [117]. During the induction of autophagy, the microtubule-associated protein light chain 3 (LC3) is proteolytically cleaved by Atg4 to generate LC3-I. LC3-I is then activated and cleaved by Atg7 and Atg3 before conjugation to phosphatidylethanolamine (PE) to generate LC3-II [118]. The Atg5-Atg12-Atg16 complex recruits LC3-II to the surfaces of the autophagosome to mediate hemifusion of membranes and selection of cargo for degradation [106]. The synthesis and processing of LC3 is increased during autophagy, making it a good indicator of autophagy levels in cells [119]. A summary of the signalling pathways regulating autophagy is illustrated in Figure 1.3 [106]. Autophagy can also be induced by unfolded protein response (UPR) under high ER stress. UPR is initiated when misfolded proteins accumulate beyond the processing capacity of the ER and serves as an alternative pathway for the removal of these proteins. In mammalian cells, double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), inositol requiring kinase 1 (IRE1) and increased cytosolic Ca2+ have been implicated as mediators of ER stress-induced autophagy [120]. It has been shown that polyglutamine repeats and other misfolded proteins that form aggregates in the cytoplasm induced ER stress [121], which induced autophagy, as observed by the upregulation of Atg12 and conversion of LC3I to LC3-II. It was demonstrated in murine cells that the PERK-elF2α signaling pathway is required for the induction of autophagy [122]. However, further work has to be done to elucidate the molecular mechanisms behind ER stress-induced autophagy. 18 ! Figure 1.3. Molecular circuitry and signalling pathways regulating autophagy. Autophagy is a complex self-degradative process that involves the following key steps: (a) control of phagophore formation by Beclin-1/VPS34 at the ER and other membranes in response to stress signalling pathways; (b) Atg5–Atg12 conjugation, interaction with Atg16L and multimerization at the phagophore; (c) LC3 processing and insertion into the extending phagophore membrane; (d) capture of random or selective targets for degradation, completion of the autophagosome accompanied by recycling of some LC3-II/ATG8 by ATG4, followed by; (e) fusion of the autophagosome with the lysosome and proteolytic degradation by lysosomal proteases of engulfed molecules. 1.4.2 Chaperone-mediated autophagy Unlike microautophagy and macroautophagy, chaperone-mediated autophagy is a selective degradation process. Substrate proteins containing the KFERQ motif are recognized by the cytosolic heat shock cognate 70 (Hsc70) protein [123]. Hsc70 forms a complex with the protein substrate, heat shock protein 40 (Hsp40) and heat shock protein 90 (Hsp90) with the aid of Hsc70-interacting protein (Hip) and Hsc70-Hsp90 organizing protein (Hop). Hsp40 activates the ATPase activity of Hsc70 and facilitates the binding of substrate proteins while Hsp90 19 ! prevents the aggregation of misfolded substrates [124]. This chaperone/substrate complex then binds to the lysosomal-associated membrane protein 2a (LAMP-2a) receptor on the lysosome membrane [124], where cytosolic Hsc70 helps to unfold the substrate protein [125]. Lysosomal Hsc70 is postulated to assist in the translocation of substrate protein into the lysosomal lumen to be degraded by lysosomal proteases [124]. 1.5 PI3K/Akt survival pathway The PI3K/Akt signaling pathway is essential for the regulation of multiple physiological processes such as cell cycle progression, transcription, translation, differentiation, apoptosis, motility and metabolism [126]. It is a major survival pathway deregulated in many cancers, contributing to cancer progression and resistance to therapy. PI3K is activated by growth factor receptor tyrosine kinases and G proteincoupled receptors, and catalyzes the production of PIP3 at the cell membrane. PIP3 in turn recruits and activates a wide range of downstream targets, including the serinethreonine kinase Akt. 3-phosphoinositide dependent protein kinase-1 (PDK1) binds to PIP3 through its pleckstrin homology (PH) domain and becomes activated. PDK1 partially activates Akt by phosphorylating at T308. Further phosphorylation at S473 results in the full activation of Akt [127]. Akt suppresses the apoptosis of cells induced by stimuli such as growth factor withdrawal, detachment of extracellular matrix components, UV irradiation, cell cycle discordance and activation of FAS signaling [128, 129]. The PI3K/Akt pathway regulates cell survival through several downstream targets. Akt downregulates either the expression or function of multiple proapoptotic 20 ! proteins which results in the enhancement of cell survival. Survival factors stimulate Akt to phosphorylate the proapoptotic Bcl-2 family member Bad, causing its sequestration from target proteins by 14-3-3 proteins [130]. Akt also inhibits the expression of Bim by phosphorylating the FOXO family of forkhead transcription factors, which are exported to the cytoplasm after binding 14-3-3 proteins [131]. In addition, it promotes the degradation of the tumour suppressor p53 by phosphorylating and activating its negative regulator Mdm2 [132]. The PI3K/Akt pathway contributes to the regulation of cell cycle progression together with the Ras/MAPK pathway. Akt phosphorylates and inhibits GSK3, thus preventing the degradation of cyclin D1 and Myc, allowing them to drive S phase entry [133, 134]. Akt-mediated phosphorylation of FOXO proteins also lifts the repression of cyclin D1 expression, as well as preventing the expression of cell cycle inhibitors [131]. Coordination between cell cycle progression and cell growth is essential for cell proliferation. Cell growth is largely regulated by protein synthesis and the high growth rate of tumour cells is likely due to the dysregulation of protein synthesis [135]. Akt activates mTOR in response to availability of nutrients and growth factors. mTOR plays a critical role in the regulation of translation initiation, ribosome biogenesis and cell growth [136]. Akt phosphorylates and inhibits tuberin, the product of the tuberous sclerosis complex-2 (TSC2), allowing Rheb-GTP to activate mTOR signalling [137]. 1.6 Project hypothesis and objectives 1.6.1 Current perspective in APL PML-RARα is widely thought to be involved in the leukemogenesis of APL. However, the exact mechanism by which this takes place remains unclear. It was 21 ! previously shown in our laboratory that the loss of function of N-CoR plays a role in the pathogenesis of APL. Further, PML-RARα has been demonstrated to bind aberrantly to N-CoR and induce its misfolded conformation, thus disrupting its tumour suppressive role in mediating the repression of self-renewal genes. Previous studies in APL mouse models have shown that transgenic mice developed an APL-like phenotype only when PML-RARα was specifically expressed in the early myeloid compartment [138, 139]. In almost all non-hematopoietic cells, PML-RARα demonstrated an anti-oncogenic effect [140] which suggests that the oncogenic potential of PML-RARα is specific to the promyelocytic compartment. In the promyelocytic compartment, its oncogenic potential is surprisingly linked to its inactivation through processing by aberrant proteases in promyelocytic cells [141] which indicates that the protein quality control mechanism UPR acts in the transformation of APL. When N-CoR is exogenously expressed with PML-RARα in human embryonic kidney 293 cells, PML-RARα binds to N-CoR and causes it to adopt a misfolded conformation and accumulate as insoluble aggregates in the ER, eventually leading to UPR-induced apoptosis [50]. However, APL cells are resistant to UPRinduced apoptosis. Recently, Ng and colleagues reported that a glycoprotein endopeptidase OSGEP, found to be selectively expressed in APL cells, cleaves NCoR [142]. Thus, ER stress generated by misfolded proteins is kept within tolerable levels, allowing APL cells to escape cell death and continue proliferating. This protease processing of N-CoR forms the basis of a defense mechanism termed as ‘late’ cytoprotective UPR, which is activated when early cytoprotective UPR comprising of molecular chaperones and ERAD fail to neutralize the toxicity of misfolded proteins 22 ! [20]. A schematic diagram of the reglation of ER stress in APL cells is shown in Figure 1.4 [20]. Further, Ng and colleagues also found genistein and curcumin to exert potent anti-proliferative effects in many APL-derived cells. Genistein has been demonstrated to inhibit N-CoR misfolding in APL cells, possibly by inhibiting the phosphorylationdependent interaction between N-CoR and PML-RARα and subsequently dissociating N-CoR from PML-RARα. This allows N-CoR to exert its effect on the differentiation of APL cells [143]. On the contrary, curcumin exers its effects by promoting the accumulation of misfolded N-CoR through inhibition of the protease-mediated degradation and ERAD. The net effect is the induction of UPR-induced apoptosis of APL cells [144]. Figure 1.4. Representation of the regulation of ER stress and UPR in APL cells. (A) In non-APL cells, accumulation of misfolded PML–RAR/N-CoR protein in the ER stimulates ER stress, which ultimately leads to UPR-induced apoptosis. (B) In APL cells, however, toxicity associated with misfolded PML–RAR/N-CoR is 23 ! neutralized owing to their processing by APL cell-specific aberrant protease activity, resulting in a reduction of ER stress and eventual protection of APL cells from UPRinduced apoptosis. The ultimate outcome of cytotoxic UPR will be antioncogenic, because it would result in elimination of PML–RAR-containing cells through apoptosis, whereas activation of cytoprotective UPR would allow the PML–RARcontaining cells to remain viable and, along with the help of additional growthpromoting stimuli, to proliferate.APL [20]: Acute promyelocytic leukemia; ER: Endoplasmic reticulum; N-CoR: Nuclear receptor corepressor; PML: Promyelocytic leukemia; RAR: Retinoic acid receptor; UPR: Unfolded protein response. 1.6.2 Hypothesis and objectives Protein misfolding is implicated in many diseases, either through the resulting disappearence of a functional protein, or accumulation of insoluble aggregates. Accumulation of misfolded proteins is a hallmark of many neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. This can lead to altered neuronal connectivity, neuronal death, high ER stress and dysfunctional ERAD machinery [145, 146]. PML-RARα and N-CoR have been reported to be misfolded and implicated in the pathogenesis of APL. However, it appears that the degradation of the misfolded proteins, rather than their accumulation, leads to cancer progression. In APL cells, insoluble aggregates of misfolded N-CoR in the Golgi have been shown to be cleaved by the APL cell-specific OSGEP protease, thus relieving ER stress and allowing APL cells to escape UPR-linked apoptosis [142]. This suggests an important role of cytoprotective UPR in the pathogenesis of APL and a possible therapeutic target. In addition, preliminary data from our laboratory has shown that APL cells, NB4, are able to survive in serum-starved conditions while non-APL cells cannot survive under the same conditions. We hypothesize that APL cells utilize misfolded N-CoR, possibly acting through cytoprotective autophagy, to provide nutrients to APL cells and confer a survival advantage (Figure 1.5). 24 ! Figure 1.5. Proposed mechanism of the effects of misfolded N-CoR in APL and non-APL cells. Artemisinin is a natural plant product extracted from Artemisia annua. Its derivatives are now used in the standard therapy for falciparum malaria worldwide [80]. Artemisinin derivatives have also shown to possess anti-proliferative effects in many cancer cell lines including leukemia [82]. Hence, it is of interest to see if artemisinin derivatives can be used for cancer treatment. The artemisinin derivatives used in this study were provided by our collaborator Professor Haynes RK from Hong Kong Science and Technology University, Hong Kong. The main objectives are: 1. To investigate if artemisinin derivatives may be a possible therapeutic drug for APL. 2. To investigate the effect of artemisinin on misfolded N-CoR in APL. 25 ! Chapter 2: Materials and Methods 2.1 Materials 2.1.1 Cell lines APL cell lines NB4, an all trans-retinoic acid (ATRA)-sensitive cell line and NB4-R1, the resistant variant, were generous gifts from Dr Homma Y (Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan) and Dr Lanotte M (INSERM U-301, Centre G. Hayem, Hôpital Saint-Louis, France) respectively. AP1060 was a kind gift from Dr Mori S (Cancer Science Institute, NUS, Singapore). Non-APL cell lines HL60, an acute myeloid leukemia (AML) cell line and K562, a chronic myelogenous leukemia (CML) cell line, were generous gifts from Dr Deng LW (Department of Biochemistry, YLL SoM, NUS, Singapore). HEK 293T cells were bought from the American Type Culture Collection (ATCC). 2.1.2 Drugs Artemisinin Different derivatives of artemisinin (Figure 2.1) were synthesized and given by our collaborator Prof Haynes RK (Hong Kong Science and Technology University, Hong Kong). The drugs were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 25 mM and aliquots were stored at 4oC. 26 ! Figure 2.1. Chemical structures of synthesized artemisinin derivatives. MG132 MG132 was obtained from Sigma Aldrich, USA and dissolved in DMSO to a stock concentration of 20 mM. Aliquots were stored at -20oC. 2.1.3 Antibodies Primary antibodies Antibody Source Company Dilution used β-actin Mouse monoclonal TU-02, Santa Cruz 1:10 000 Akt Rabbit polyclonal Cell Signaling 1:1000 Beclin-1 Rabbit polyclonal Cell Signaling 1:1000 Caspase 3 Rabbit polyclonal Cell Signaling 1:1000 Caspase 4 Rabbit polyclonal Cell Signaling 1:1000 Caspase 8 Mouse monoclonal 1C12, Cell Signaling 1:2000 27 ! Antibody Source Company Dilution used Caspase 9 Rabbit polyclonal Cell Signaling 1:1000 Flag Mouse monoclonal F3165 clone M2, 1:10 000 Sigma HA Rabbit polyclonal Y11, Santa Cruz 1:1000 LC3 Rabbit polyclonal NB100-2220, 1:3000 Novus Biologicals MTOR Rabbit polyclonal Cell Signaling 1:1000 N-CoR Goat polyclonal C-20, Santa Cruz 1:500 PARP Rabbit polyclonal Cell Signaling 1:1000 Phospho-Akt Mouse monoclonal T308, Cell Signaling 1:1000 Phospho-MTOR Rabbit polyclonal Ser2448, 1:1000 Cell Signaling PI3K Class III Rabbit polyclonal Cell signaling 1:1000 RAR Rabbit polyclonal C20, Santa Cruz 1:1000 Survivin Rabbit monoclonal Cell Signaling 1:1000 Secondary antibodies Antibody Company Goat anti-mouse IgG (H+L), HRP Zymed Laboratories Dilution Used 1:10 000 Zymed Laboratories 1:10 000 Zymed Laboratories 1:10 000 conjugate Goat anti-rabbit IgG (H+L), HRP conjugate Mouse anti-goat IgG (H+L), HRP conjugate 28 ! 2.1.4 Primers Semi-quantitative RT-PCR primers Gene Sequence B2M F: 5’-ATCCAGCGTACTCCAAAGAT-3’ Annealing temperature (oC) 58 Number of cycles 60 30 30 R: 5’-TTACATGTCTCGATCCCACT-3’ N-CoR F: 5’-TACCGCAGGAGCCATACAAGA-3’ R: 5’-GCTCAGTTGTGCTTTGGGAGC-3’ 2.2 Methods 2.2.1 Cell culture All leukemic cell lines were maintained in Rosewell Park Memorial Institute 1640 (RPMI) medium (Invitrogen,USA) supplemented with 10% fetal bovine serum (FBS), purchased from Hyclone, UT. The human embryonic kidney (HEK) 293T cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM), purchased from Sigma, USA, supplemented with 10% FBS. All cells were cultured at 37oC in a humidified atmosphere with 5% CO2. 2.2.2 Transfection 293T cells were seeded at a density of 200x104 cells per 10-cm plate or 20x104 cells per 6-well plate. Transfection was carried out 18-22 hrs after seeding. Fugene 6 (Roche) was added to serum free DMEM and incubated for 5 mins at room temperature. DNA is then added to the mixture and incubated for another 15 mins at room temperature. The mixture was then slowly added dropwise to the cells. The cells were harvested 48 hrs after transfection. 29 ! Size of plate Fugene (µL) SF-DMEM (µL) 6-well 6 94 Maximum DNA (µg) 2 10 cm 18 382 6 2.2.3 Cell proliferation assay The cell proliferation assay was performed using the Cell Proliferation Kit I (MTT) (Roche) according to manufacturer’s instructions. Cells were seeded into 96well plates at 4000 cells/well in 100 µL culture medium with the respective drug concentrations. The cells were incubated at 37oC with 5% CO2 for 24-96 hrs. After the incubation period, 10 µL of the MTT labeling reagent was added to each well and incubated at 37oC for 4 hrs. Next, 100 µL of the solubilisation reagent was added to each well and allowed to stand in the incubator overnight for complete solubilisation of the purple formazan crystals. A microplate reader (Ultramark Microplate Imaging System, Biorad) was used to measure the spectrophometrical absorbance of the samples at a wavelength of 595 nm and a reference wavelength of 655 nm. 2.2.4 Cell lysis for protein extraction Leukemic suspension cells were centrifuged at 200g for 5 mins and the pellet was washed twice with ice-cold 1x PBS. Harvesting of adherent cells was done by first removing the cell culture media and cells were washed with ice-cold 1x PBS. Cells were trypsinized and cell culture media was added to neutralize the trypsin. Cells were centrifuged at 200g for 5 mins and the pellet was washed twice with icecold 1x PBS. 4 times the cell pellet volume of 1x SDS sample buffer (4x buffer250mM Tris-HCl (pH 6.8), 40% glycerol, 9.2% SDS, 0.01% bromophenol blue, 20% β-mercaptoethanol added fresh prior use; dilute with dH2O to get 1x) was added to 30 ! lyse the cells. The cells were sonicated two times on ice, 10 s each, with the sonicator (Branson Sonifier 150), output power of 5 W. The protein samples were then heated at 50oC for 10 mins and lysates were stored at -80oC. 2.2.5 Western blotting Proteins in the cell lysates were seperated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using resolving gels of 6-15% and 5% stacking gel. Electrophoresis was carried out using 1x cold running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 4oC. After electrophoresis, the proteins on the gel was transferred onto a pre-wet PVDF membrane using a wet transelectroblotting system (Bio-Rad Inc., England) at a constant current of 75 mA for 2 hrs at 4oC in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 10% or 20% methanol). 10% methanol was used except when proteins of interest are below 30 kDa, where 20% methanol was used. After transfer, the membrane was blocked in PBS-T (PBS with 0.1% Tween-20) containing 5% milk for at least 1 hr at room temperature. To detect phosphorylated proteins, the membrane was blocked in TBS-T (TBS with 0.1% Tween-20) containing 5% BSA. The membrane was then incubated with primary antibodies against the proteins of interest in the blocking buffers overnight at 4oC. After washing 3 times in PBS-T (or TBS-T, depending on blocking buffer) for 10 mins each to remove excess antibodies, the membrane was incubated with the appropriate HRP-conjugated secondary antibodies in blocking buffer for 1 hr at room temperature. Excess antibodies were washed away by washing with PBS-T (or TBS-T) for 5 times, 10 mins each. The immunoreactive bands were detected by the Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer) using an X-ray film, which was developed by a Konica Minolta SRX-101A film processor. 31 ! 2.2.6 Polyacrylamide gels Resolving gel Stacking gel Components 6% 8% 10% 12% 15% 5% Water (mL) 5.3 4.6 4.0 3.3 2.3 6.8 30% acrylamide 2.0 2.7 3.3 4.0 5.0 1.71 1.5M Tris pH 8.8 2.5 2.5 2.5 2.5 2.5 -- -- -- -- -- -- 1.25 10% SDS (mL) 0.1 0.1 0.1 0.1 0.1 0.1 10% APS (mL) 0.1 0.1 0.1 0.1 0.1 0.1 TEMED (µL) 8 6 4 4 4 10 Total volume 10 10 10 10 10 10 mix (mL) (mL) 1M Tris pH 6.8 (mL) (mL) 2.2.7 Coomassie staining Cell lysates were loaded into gels and resolved by SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Blue staining solution (0.25% Coomassie Blue, 10% acetic acid, 50% methanol) for 30 mins. The stained gel was then destained overnight with Coomassie destaining solution (10% acetic acid, 50% methanol). Coomassie gels were used to check the integrity of protein samples and as a loading control to ensure equal loading when running SDS-PAGE for proteins of interest. 32 ! 2.2.8 RNA extraction NB4 cells were pelleted by centrifugation at 200g for 5 mins at 4oC, then washed with ice cold 1x PBS. Purification of total RNA was carried out using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The cells were lysed in Buffer RLT (10 µl β-mercaptoethanol was added per 1 ml Buffer RLT before use) and homogenized using needle and syringe. 1 volume of 70% ethanol was added to the homogenized lysate and the sample was loaded onto the RNeasy Mini Spin column, followed by centrifugation at 8000 g for 15 s. The column was washed with Buffers RW1 and RPE. Lastly, purified RNA was eluted with 50 µl RNase-free water and its concentration was determined using Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA). 2.2.9 Reverse-transcription polymerase chain reaction (RT-PCR) First, 3 µg of the purified RNA template was used for cDNA synthesis using the RT-PCR System kit (Promega, USA). Reverse transcription was performed at 42oC for 1 hr, and next at 65oC for 15 mins. Synthesis was terminated by incubation at 95oC for 5 mins. Next, PCR amplification was carried out using the Thermal Cycler GeneAmp®PCR System 9600 (Applied Biosystems, USA). The annealing temperature and number of cycles were adjusted according to the primers used. PCR amplification was performed according to the steps listed in Table 2.1. 33 ! Table 2.1. Steps for PCR amplification Temperature (oC) Time 1. Initialization 94 5 mins 2. Denaturation 94 30 s 3. Annealing 60 30 s 4. Elongation 72 30 s 5. Final elongation 72 7 mins 6. Final hold 4 ∞ Steps Cycle steps 2-4 for 30 times 2.2.10 Flow cytometry apoptosis assay The Annexin-V FITC Apoptosis Detection Kit I (BD Bioscience) was used for the detection of apoptosis by flow cytometry. The assay was carried out according to the manufacturer’s instructions. NB4 cells were seeded at 4x104 cells/ml in a 6-well plate and 1 uM of artemisinin derivative 11; henceforth referred to as GC011; was added for the required treatment time before harvesting. The cells were washed twice with ice-cold 1x PBS and resuspended in 1x binding buffer to a concentration of 10x106 cells/ml. 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) were added to 100 µl of the resuspended cells (1x105 cells). The cells were vortexed gently and incubated for 15 mins at room temperature in the dark. 400 µl of 1x binding buffer was added the the cells and analyzed by flow cytometry. The untreated controls – unstained cells, cells stained with only Annexin V and cells stained with only PI were used to set up the compensation and quadrants. Flow cytometry analysis was carried out on the BDTM LSR II flow cytometer using the BD FACSDivaTM software. 34 ! 2.2.11 Proteasome sensor assay 20x104 293T cells were seeded in 6-well plates and each well was transfected with 2 µg of the proteasome sensor vector pZsProSensor-1 (BD Biosciences, Clonetech) as described in Section 2.2.2. The vector contains the mouse decarboxylase degradation domain (MODC d410) fused to the C-terminus of ZsGreen, a green fluosescent protein. MODC d410 fusion proteins are targeted to the proteasome and can be used to monitor proteasome activity. 24 hrs after transfection, cells were treated with 0 µM and 1 µM of GC011. 24 hrs after treatment, cells were viewed using the Nikon Eclipse TE2000-S microscope. 2.2.12 Immunostaining and fluorescence microscopy 20x104 293T cells were seeded in 6-well plates. Cells were co-transfected with 1 µg of pACT-N-CoR-Flag and 1 µg of pACT-PML-RARα-HA as described in Section 2.2.2, and treated with varying concentrations of GC011 for 24 hrs. As the treated cells were found to detach after fixing, the cells were trypsinized and cytospun onto glass slides using the Shandon Cytospin 4 machine before fixing with 4% paraformaldehyde in PBS for 30 mins at 37oC. After washing 3 times, 5 mins with PBS, cells were permeabilized using 0.2% Triton X-100 in PBS for 5 mins on ice. The cells were washed 3 times with PBS and blocked with 5% BSA in PBS for 30 mins at room temperature. After a brief rinse, the cells were incubated with anti-Flag and anti-HA primary antibodies at a dilution of 1:200 in 5% BSA for 3 hrs at room temperature. The cells were washed thrice again with PBS and incubated with appropriate Alexa Fluor secondary antibodies (Molecular Probes, Eugene, OR) at a dilution of 1:200 in 5% BSA for 1 hr at room temperature. After washing 3 times with PBS, the cells were stained with 150 nM of 4, 6-diamidino-2-phenylindole (DAPI) in 35 ! 5% BSA for 5 mins, washed thrice with PBS and briefly rinsed with water. The slides were mounted with SlowFade® Gold anti-fade reagent (Molecular Probes, CA) and sealed with nail polish. The slides were viewed using the Nikon eclipse TE2000S microscope and images were captured and analyzed using Nikon ACT-2U imaging software. 2.2.13 Measurement of internal ATP levels The ATP Bioluminescence Assay Kit HS II (Roche) was used to measure internal ATP levels of cells and the assay was carried out according to the manufacturer’s instructions. Cells were treated with 1 µM of GC011 for 0-24 hrs for the assay. 1x105 live cells were counted after staining with Tryphan blue, pelleted and washed twice with ice cold 1x PBS. The cells were then resuspended to a concentration of 1x105 cells/ml with dilution buffer. 300 µl of cell lysis buffer was added to 300 µl of the cell suspension and incubated for 5 mins at room temperature. The cell lysate was then centrifuged at 10 000 g for 1 min at 4oC and the supernatant was retained. The supernatant was diluted appropriately with dilution buffer. 100 µl of luciferase reagent was added to 50 µl of the diluted sample in a luciferase tube, mixed and luminescence was immediately measured using the Sirius Single Tube Luminometer (Berthold Detection System). Measurement was started after a 1 s delay and integrated for 10 s. Each sample was assayed in triplicates. 36 ! Chapter 3: Results 3.1 Artemisinin selectively inhibits the growth of APL but not non-APL cells Artemisinin has been shown to have anti-proliferative properties in various cancers but due to its poor solubility and bioavailability, many derivatives have since been developed. Our collaborator Professor Haynes RK has synthesized eight different artemisinin derivatives (Figure 2.1) and the primary objective is to identify if there is a derivative effective against APL induced by N-CoR misfolding. The therapeutic potential of these derivatives was tested in the APL cell line, NB4, via MTT cell proliferation assays. Using a range of concentrations between 0 µM to 25 µM, the derivatives inhibited the proliferation of NB4 in a dose-dependent manner (Figures 3.1). Artemisinin derivatives 9 and 11 were found to be among the most potent (Figure 3.1F and H), while derivatives 1 and 6 did not show any antiproliferative effect (Figure 3.1A and E). Next, derivative 11 (henceforth referred to as GC011), shown to be the most effective, was selected to be further tested in two other APL cell lines, NB4-R1 and AP1060 (Figure 3.2A-C). GC011 also inhibited the proliferation of NB4-R1, a RA-resistant cell line. AP1060 is resistant to both RA and ATO [147]. Hence, GC011 is effective in both RA-sensitive and resistant cells. The effective concentration of GC011 in AP1060 is 1 µM, which is the highest among the three APL cell lines. The sensitivity of two other non-APL leukemic cell lines (HL60 and K562) to the different artemisinin derivatives was also tested. Our findings suggest that artemisinin specifically inhibits the proliferation of APL cells but not non-APL cells (Figure 3.3A-B). Further, the selective sensitivity of NB4 is especially evident at a higher concentration of 5 µM. 37 ! A B C 38 ! D E F 39 ! G H Figure 3.1. Artemisinin derivatives inhibit proliferation of NB4 cells. NB4 cells were treated with various concentrations of the different artemisinin derivatives for the durations indicated. Growth of the treated cells was measured by the MTT assay. (A and E) GC001 and GC006 failed to induce any growth inhibition. (B-D, F-H) GC002 and GC004 are effective at 5 µM; while the rest are effective at 1 µM. (F and H) GC009 and GC011 are the most effective in inhibiting NB4 cell proliferation. Triplicates were measured for each treatment and time-point. 40 ! A B C Figure 3.2. GC011 inhibits cell proliferation of RA-sensitive and RA-resistant APL cell lines. (A) RA-sensitive NB4, (B) RA-resistant NB4-R1 and (C) RAresistant AP1060 cells were treated with various concentrations of GC011 as stated for the durations as indicated. GC011 is equally effective in both NB4 and NB4-R1. The effective concentration in AP1060 is higher at 5 µM. Triplicates were measured for each treatment and time-point. 41 ! A B Figure 3.3. Artemisinin derivatives selectively inhibit proliferation of APL cells. APL cells NB4 and non APL cells HL60 and K562 were treated with (A) 1 µM and (B) 5 µM of the various artemisinin derivatives for 72 hrs. Growth kinetics of the treated cells was measured by MTT assay. Growth inhibition was most prominent in NB4 cells, while K562 cells were the most resistant. Triplicates were measured for each treatment and time-point. 42 ! 3.2 Artemisinin derivative, GC011, promotes apoptosis of APL cells NB4 cells showed different morphological features characteristic of apoptosis such as membrane blebbing, cell shrinkage and cell fragmentation after exposure to GC011. We observed varying combinations and degrees of apoptotic features from 48 to 72 hrs of treatment. To determine if there was an activation of apoptosis, AnnexinV apoptosis assay using the flow cytometry was performed. Treatment with 1 µM GC011 induced an increase in the proportion of Annexin-V positive NB4 cells over time (Figure 3.4). Annexin-V positive cells have exposed phosphatidylserine which are indicative of apoptotic cells. GC011 also induced the activation of apoptotic markers. Cleavage of the initiator caspase 8, effector caspase 3, and PARP occurred after 24 hrs of treatment (Figure 3.5). Cleavage of caspase 9, on the other hand, occurred after 48 hrs of treatment. These results collectively suggest that GC011 trigger apoptosis in APL cells. Figure 3.4. GC011 induces apoptosis in NB4 cells. NB4 cells were treated with 1 µM GC011 for the durations as indicated. The population of Annexin V-positive cells indicates apoptotic cells, which increases with the time of treatment. 43 ! Figure 3.5. GC011 activates the apoptotic pathway in NB4 cells. NB4 cells were treated with 1 µM GC011 for 24 and 48 hrs. Western blot analysis was carried out on the crude lysates using anti-caspase 8, anti-caspase 9, anti-caspase 3 and anti PARP antibodies. GC011 induced the cleavage of the above apoptotic proteins from 24 hrs. 44 ! 3.3 Artemisinin derivative, GC011, promotes the degradation of misfolded NCoR and PML-RARα It has been shown previously that misfolded N-CoR and PML-RARα are cleaved in NB4 cells [141, 142]. Drugs such as AEBSF and curcumin were found to inhibit the growth of APL cells by inhibiting the degradation of misfolded N-CoR [142, 144]. This led to an accumulation of misfolded proteins and induction of UPRinduced apoptosis. To investigate if artemisinin has a similar effect on the levels of NCoR in NB4, Western blot assay was done on NB4 cells treated with GC004, GC006 and GC011. Based on the proliferation assay, GC011 exerted the greatest antiproliferative effect, while GC006 had no effect on cell proliferation (Figure 3.1 E and H). GC004, on the other hand, possessed a weaker anti-proliferative effect than GC011 (Figure 3.1 D). In this assay, GC006 was used as a control. We expected stabilization of full-length N-CoR in NB4 cells after treatment with GC011. However, we observed that N-CoR was instead further degraded after treatment. Full-length NCoR was clearly reduced and the cleaved band was completely abolished after treatment (Figure 3.6). This observation suggests that GC011 degrades both fulllength and cleaved N-CoR. The extent of degradation observed with each artemisinin derivative compound corresponded to the degrees of growth inhibition exerted. At 1 µM concentration, GC006, which had no effect on the growth inhibition of NB4, failed to induce degradation of N-CoR while GC004 induced a slight decrease in the amount of full-length and cleaved N-CoR. Next, we wanted to ascertain if the decrease in N-CoR levels seen is due to GC011 and not by proteases inherent in APL cells. N-CoR was transfected together with PML-RARα to induce misfolding and then treated with GC011 in 293T cells. Treatment time was shortened to 24 hrs as 293T cells were more sensitive to the drug 45 ! GC011. N-CoR and PML-RARα proteins were found decreased in a dose-dependent manner after treatment (Figure 3.7). Imunofluorescence analysis was then employed to validate the result observed. Transfected N-CoR and PML-RARα was seen to colocalize in the cytoplasm in 293T cells. Treatment with GC011 resulted in a decrease in frequency and intensity of N-CoR signal (Figure 3.8). These results collectively indicate that GC011inhibits the degradation of misfolded N-CoR. To test if GC011-induced degradation of N-CoR is selective only in APL cells, N-CoR levels in the non-APL cell line HL60 was compared with NB4 after treatment. After 48 hrs, N-CoR in NB4 was completely degraded while there was a marginal decrease in HL60 (Figure 3.9). This data suggests that GC011 is relatively selective for APL cells, as previously demonstrated by cell proliferation assays (Figure 3.3). Figure 3.6. GC011 enhanced the degradation of N-CoR and PML-RARα in NB4 cells. NB4 cells were treated with 1 and 5 µM of the artemisinin derivatives as indicated for 48 hrs. Levels of N-CoR and PML-RARα were detected by western blotting using their respective antibodies. DMSO and GC006 treated cells were used as positives control for N-CoR and PML-RARα detection. Β-actin served as a loading control. 46 ! Figure 3.7. GC011 induced the degradation of transfected N-CoR and PMLRARα in 293T cells in a dose-dependent manner. 293T cells were transfected with N-CoR and PML-RARα plasmids and treated with various concentrations of GC011 for 24 hrs. Western blot analysis of N-CoR and PML-RARα was done using anti-Flag and anti-HA antibodies respectively. Coomassie blue staining was done for loading control. 47 ! DAPI% % % 0%µM% % % 0.5%µM% % % 1%µM% N'CoR% PML'RARα% Merge% 48 ! DAPI% N'CoR% PML'RARα% Merge% 2.5%µM% 5%µM% Figure 3.8. GC011 reduced the expression of transfected N-CoR and PML-RARα in the cytosol of 293T cells in a dose-dependent manner. 293T cells were transfected N-CoR and PML-RARα plasmids and treated with various concentrations of GC011 for 24 hrs. Cells were stained with anti-Flag to visualize N-CoR (green) and anti-HA to visualize PML-RARα (red). DNA was stained with DAPI (blue). Signals were acquired with fluorescent microscopy. N-CoR and PML-RARα were localized to the cytosol and their expression decreased with treatment. 49 ! Figure 3.9. GC011 induced a significant down-regulation of N-CoR in APL cells but not non-APL cells. APL (NB4) and non-APL (HL60) cells were treated with 1 µM GC011 for 24 and 48 hrs. N-CoR in crude lysates was detected by Western blot analysis. 3.4 Degradation of N-CoR by GC011 via the proteasome-dependent pathway in APL cells. 3.4.1 GC011-induced N-CoR degradation is mediated via the proteasome pathway To investigate the mechanism of GC011-induced degradation of N-CoR, mRNA levels of N-CoR in treated and untreated cells was first measured via semiquantitative PCR. As shown in Figure 3.10, treatment with GC011 resulted in a 50 ! marginal decrease in the mRNA level of N-CoR. The continued presence of N-CoR mRNA could not account for the total degradation of N-CoR protein observed (Figure 3.6) and this suggests that GC011 has a post-translational effect on N-CoR. One of the main routes for protein degradation in the cell is via the proteasome. It has been proposed that artemisinin induced the ubiquitination and proteasomal degradation of the androgen receptor in prostate cancer cells [77, 148]. Previous results from our laboratory also showed that N-CoR in the presence of PML-RARα becomes heavily ubiquitinated in NB4 cells [50]. To test if the decrease in N-CoR levels was a result of an increased proteasomal activity by GC011, the proteasome activity was determined using the proteasome sensor vector pZsProSensor-1 in GC011 treated 293T cells. The vector contains the ZsGreen fluorescent protein fused to mouse ornithine decarboxylase degradation domain (MODC d410), which targets it to the proteasome. Thus, the intensity of fluorescent signal is inversely proportional to proteasome activity. The number of cells with the green fluorescence signal and signal intensity decreases with increasing concentrations of GC011, indicating that GC011 enhances proteasomal activity in a dose-dependent manner (Figure 3.11). Figure 3.10. GC011 did not cause significant change to mRNA levels of N-CoR in NB4 cells. NB4 cells were treated with 1 µM GC011 for 24 and 48 hrs. Transcript levels of N-CoR were analyzed by semi-quantitative PCR. B2M was used as a loading control. 51 ! N"CoR"GFP) Phase)contrast) Merge) ) ) 0) µM) ) ) 0.5) µM) ) ) 1) µM) ) ) 2.5) µM) ) ) 5) µM) Figure 3.11. GC011 enhanced the degradation of the proteasome sensor in 293T cells in a dose-dependent manner. 293T cells were transfected with the proteasome sensor and treated with various concentrations of GC011 for 24 hrs. Green fluorescence of the proteasome sensor was visualized. GC011 reduced the intensity of the signal, indicating an increased proteasome activity. ! ! 52 ! 3.4.2 N-CoR is rescued by MG132 in GC011-treated APL cells To further verify if GC011-induced degradation of N-CoR is proteasome dependent, MG132 was used to determine if N-CoR can be stabilized. MG132 is a specific 26S proteasome inhibitor known to stabilize ubiquitinated proteins. NB4 cells were first treated with 1 µM GC011 for 24 hrs and varying concentrations of MG132 was added 12 hrs before harvesting. Western blot analysis showed that both full length and cleaved N-CoR protein were stabilized with 10 µM MG132 (Figure 3.12). However, MG132 failed to prevent the down-regulation of PML-RARα. To confirm the dependence on the proteasome for N-CoR degradation, 293T cells were transfected with N-CoR and PML-RARα and treated with 2.5 µM GC011 and MG132 in the same manner described for NB4 cells. Similar to the observation in NB4, MG132 treatment of transfected 293T cells resulted in stabilization of N-CoR protein in a dose-dependent manner. Maximum stabilization was obtained at 20 µM MG132. Likewise, there was no up-regulation of PML-RARα in 293T cells after MG132 treatment (Figure 3.13). 53 ! Figure 3.12. MG132 reversed GC011-induced N-CoR degradation in NB4 cells. NB4 cells were treated with 1 µM GC011 for 24 hrs. 12 hrs before harvest, various concentrations of MG132 was added. N-CoR and PML-RARα levels were detected by Western blotting. Only N-CoR was stabilized by MG132. 54 ! Figure 3.13. MG132 reversed GC011-induced N-CoR degradation in 293T cells. 293T cells were transfected with N-CoR and PML-RARα plasmids and treated with 2.5 µM GC011 for 24 hrs. 12 hrs before harvest, various concentrations of MG132 was added. N-CoR and PML-RARα levels were detected by Western blotting. Only N-CoR was stabilized by MG132. 3.5 Autophagy is blocked by GC011 in APL cells 3.5.1 Autophagy is activated in APL cells and contributes to cellular growth Cleavage of N-CoR in APL cells protects the cells from the cytotoxic effects of UPR and confers a survival and growth advantage [142]. We speculate that the protective arm of autophagy known as cytoprotective autophagy, may be a part of the proteolytic mechanism regulating this phenomenon. Autophagy is a cellular process activated in response to stress conditions such as nutrient deprivation and ER stress. In tumour cells, it has been reported that autophagy is induced to meet the high 55 ! metabolic demands required for increased cell proliferation [105]. When there is an accumulation of misfolded proteins in cells, UPR is activated and followed by cessation of protein synthesis, resulting in lower intracellular energy levels. Thus it is likely that in APL cells, degradation of misfolded N-CoR is a result of autophagy activation to compensate lower energy levels. To determine if autophagy is activated in NB4, LC3-II and LC3-I was measured in NB4, HL60 and K562 cells. LC3-I is processed to LC3-II and can be found on the membranes of autophagosomes during autophagy. Hence, an increased LC3-II/LC3-I ratio may indicate an increased activation of autophagy. We did not observe any significant difference between the levels of LC3-I and LC3-II in nonAPL cells, HL60 and K562. In these non-APL cells, basal level of autophagy, which is a common characteristic in all mammalian cells, was detected. However, in APL NB4 cells, there was a significantly higher level of LC3-II as compared to LC3-I (Figure 3.14). These results indicate that the level of autophagy is higher in APL cells compared to non-APL cells. To test the hypothesis that cytoprotective autophagy is activated in APL cells to compensate for the reduced intracellular energy levels arising from reduced protein synthesis, the ATP levels in NB4 cells were determined after bafilomycin A1 (BA-1) treatment. BA-1 blocks the fusion of autophagosomes and lysosomes during autophagy [149]. As shown in Figure 3.15, BA-1 treatment resulted in a dosedependent decrease of ATP levels in NB4 cells. Next, we seek to investigate if the activation of cytoprotective autophagy confers a survival advantage and contributes to the growth of APL cells under stress conditions. Both APL and non-APL cells were subjected to glucose starvation. When grown in glucose-free media, NB4 cells continued to grow, though at a slower rate 56 ! compared to growth in normal media (Figure 3.16A). In contrast, the growth of HL60 and K562 ceased when grown in glucose-free conditions (Figure 3.16B). These results collectively suggest that activation of cytoprotective autophagy confers a prosurvival advantage to APL cells. Figure 3.14. LC3-II/LC3-I ratio is high in APL cells. Levels of LC3 in APL and non-APL cells were determined through Western blotting with anti-LC3 antibody. Cells were incubated with lysosomal protease inhibitors, E64D and pepstatin A for 5 hrs to partially inhibit the degradation of LC3-II. This work was done by Ng APP. 57 ! Figure 3.15. BA-1 reduces the intracellular ATP level in NB4 cells in a dosedependent manner. NB4 cells were treated with various concentrations of BA-1 for 48 hrs and intracellular ATP levels were measured by ATP assay. This work was done by Ng APP. 58 ! A B Figure 3.16. APL cells are resistant to glucose starvation-induced growth inhibition. APL (NB4) and non-APL (HL60 and K562) cells were grown under normal conditions (A) and under glucose-free conditions (B). All 3 cell lines grew at a similar rate when cultured in nutrient medium. When cultured in glucose-free medium, only NB4 continued to grow, but at a slower rate. Growth of non-APL cells was completely inhibited. This work was done by Ng APP. 59 ! 3.5.2 GC011 blocks autophagy in NB4 cells Since degradation of misfolded N-CoR is speculated to provide intracellular energy through autophagy and support the survival of APL cells, it can be expected that further degradation of N-CoR by artemisinin can further activate autophagy and enhance cellular growth. However, we observed that artemisinin treatment induced growth inhibition and apoptosis of APL cells. To elucidate the mechanism of action exerted by GC011, its effect on autophagy in APL cells was examined. Western blot analysis was carried out on several autophagic markers such as class III PI3K, Beclin1 and LC3. These proteins are necessary for phagophore formation [108]. GC011 treatment resulted in the down-regulation of all three proteins (Figure 3.17) and the down–regulation was more evident at 48 hrs. This finding suggests that GC011 inhibits early autophagy in APL cells. Figure 3.17. GC011 inhibits autophagy in NB4 cells. NB4 cells were treated with 1 µM GC011 for 24 and 48 hrs. Western blot analysis was carried out on crude lysates using anti-class III PI3K, anti-Beclin-1 and anti-LC3. 60 ! 3.5.3 GC011-induced degradation of N-CoR is associated with a decrease in intracellular energy. It has been shown that inhibition of cytoprotective autophagy with BA-1 results in the reduction of intracellular ATP levels in NB4 cells (Figure 3.15). As observed in the results above, GC011 also exerted an inhibitory effect on autophagy (Figure 3.17). Thus, we wanted to examine if GC011 could also have a similar effect as BA-1 on intracellular energy levels. First, N-CoR levels after various time-point treatments with GC011 were determined by Western blot analysis. Concurrently, the intracellular ATP levels at the various time-points were also measured. Consistent with previous results, treatment with GC011 led to the degradation of N-CoR. Prominent degradation could be seen at 18 hrs, with maximum degradation at 24 hrs (Figure 3.18A). Corresponding to the decreasing N-CoR levels, we observed decreasing intracellular ATP levels in NB4 cells with increasing exposure time to GC011 (Figure 3.18B). This finding suggests that the catabolism of misfolded N-CoR through autophagy can act as a source of ATP generation in NB4 cells. 61 ! A B Figure 3.18. Reduction of intracellular ATP levels is associated with GC011induced N-CoR degradation in NB4 cells. NB4 cells were treated with 1 µM GC011 for the durations as indicated. (A) N-CoR levels were detected by Western blotting assay. (B) Intracellular ATP levels were detected by ATP assay. The decrease in ATP levels corresponded with the decrease in N-CoR levels. 62 ! 3.6 GC011 blocks autophagy via the PI3K/Akt survival pathway in APL cells The PI3K/Akt signaling pathway regulates several important cellular processes such as cell cycle progression, metabolism and apoptosis that are implicated in carcinogenesis [126]. Activation of this pathway results in uncontrolled cell growth and survival, conferring a growth advantage to tumour cells. mTOR, a protein downstream of Akt, is a main regulator of autophagy and protein synthesis [150]. A previous report by Ren et al. suggested that the PI3K pathway played an important role in the inhibition of autophagy and induced apoptosis in selenium-treated NB4 cells [151]. Since treatment with GC011 has shown to promote apoptosis and inhibit autophagy, we determine to examine if GC011 has a regulatory effect on the PI3K/Akt pathway. As expected, we observed a down-regulation of the phosphorylation and activation of Akt and mTOR in NB4 cells after 48 hrs exposure to GC011 (Figure 3.19). The level of survivin after treatment was also determined. Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is constitutively expressed in most cancers [152]. It can inhibit apoptosis by interfering with mitochondrial cytochrome c release and caspase-9 activity [153]. Since we have observed an activation of caspase-9 after treatment, we twanted to determine if there was a corresponding decrease in survivin. Indeed, GC011 also led to a downregulation of survivin protein (Figure 3.19). Together, these results suggest that the decreased survival of NB4 cells after GC011 treatment may be mediated by the PI3K/Akt pathway. 63 ! Figure 3.19. GC011 inhibits the PI3K/Akt pathway in NB4 cells. NB4 cells were treated with 1 µM GC011 for 24 and 48 hrs. Western blot analysis was carried out on crude lysates using anti-Akt, anti-phospho Akt, anti-mTOR, anti-phospho mTOR and survivin. 64 ! Chapter 4: Discussion 4.1 Artemisinin shows promise as a therapeutic agent in APL Artemisinin, the active component of the Artemisia annua plant, has been used in traditional Chinese medicine for more than 2000 years to treat various illnesses [77]. Currently, artemisinin and its bioactive derivatives such as artemether, artesunate and dihydroartemisinin are recommended by the World Health Organization (WHO) for the treatment of multidrug-resistant strains of malaria [154]. Of great interest, artemisinin derivatives exhibit potent anti-cancer activity in a variety of cancer cells. An analysis of 55 human cancer cell lines by the Developmental Program of the National Cancer Institute, USA showed that artesunate was most active against leukemic and colon cancer cell lines [82]. The mean IC50 in leukemic cell lines was reported to be about 1.11±0.56 µM, which was similar to the results from this study. In this study, treatment with 1 µM of GC011 for 72 hrs resulted in about 80% growth inhibition in NB4 and NB4-R1 cells, and approximately 50% in AP1060 cells. This growth inhibition was seen more pronounced in APL cells than non-APL cell lines, demonstrating a higher selectivity for APL cells. APL cell lines exhibited response to GC011 at concentrations of 1 µM or lower, while growth inhibition was only observed in non-APL cells at 5 µM. Artemisinin can also mediate its anti-proliferative effect through many cellular pathways, such as activation of apoptosis, cell cycle arrest, and inhibition of angiogenesis and cell migration [77]. Together, artemisinin and its derivatives are attractive therapeutic agents for cancer treatment. It is a natural product and is currently in use worldwide for the treatment of malaria. Thus, toxicity problems normally associated with developing drugs may be circumvented. Previous studies have also shown that the cytotoxic effects of artemisinin derivatives are selective for cancer cells and have much less 65 ! effect on neighbouring normal cells [155, 156]. A plausible explanation for the observation is that cancer cells contain relatively higher concentrations of iron, which cleave the endoperoxide bridge in artemisinin to release free radicals [157]. Taken together, the relatively higher selectivity of GC011 for APL cells may indicate that GC011 can be a potential therapeutic agent for APL. 4.2 GC011 induces caspase-activated apoptotic pathways in APL Artemisinin may mediate apoptosis through activation of apoptotic regulators, upregulation of p38 mitogen-activated protein kinase (MAPK) and p53-mediated apoptosis and production of reactive oxygen species (ROS) [77]. Apoptotic assays carried out with Annexin V staining showed that GC011-treated APL cells undergo apoptosis. Consistent with reports in other cancers [158, 159], GC011 induced the activation of caspases-3 and -9. The caspase-9 apoptotic pathway mediates intrinsic apoptotic signals and is activated when there is increased cytochrome c release from the mitochondria [160]. An increase in cytochrome c release may be due to increased ER stress after treatment which activates the IRE1 pathway and Bim phosphorylation [161]. Stockwin et al. has reported that artemisinin dimmers upregulates the expression of ER stress markers in 2 different cancer cells [162]. Thus, it may be possible that GC011 can induce ER stress, which may be a factor for caspase-9 activation in GC011-treated cells. Additionally, GC011 also induced the activation of caspase-8. It is possible that there is cross-talk between the caspase-8/Bid pathway and the caspase-9 mitochondrial pathway in GC011-induced apoptosis. Similar observations were also made by Gu et al. on aspirin-induced apoptosis in gastric cancer cells [163]. The Bcl-2 family member Bid provides a link between these two pathways. Bid is cleaved by caspase-8 and its truncated form tBid translocates to the 66 ! mitochondria where it acts together with Bax and Bak to induce cytochrome c release to activate the caspase-9 pathway [164]. However, further investigation is required to validate this theory. 4.3 GC011 enhances N-CoR degradation through the proteasome pathway In APL cells, misfolded N-CoR is cleaved by a glycopeptidase, alleviating ER stress and this allows cells to escape UPR-induced apoptosis [142]. Previously, our laboratory discovered that therapeutic agents such as AEBSF and curcumin inhibited the degradation of N-CoR. Accumulation of misfolded proteins led to an in increase in ER stress levels and induction of UPR-induced apoptosis [142, 144]. In this study, we observed an increase in ER stress and apoptosis in GC011-treated NB4 cells which indicated that GC011 could also inhibit N-CoR degradation in a similar fashion. Unexpectedly, N-CoR and PML-RARα were found to be further degraded after treatment, suggesting a reactivation of a cellular protein quality control mechanism by artemisinin. Intracellular accumulation of misfolded proteins tends to inhibit the cellular protein quality control mechanism regulated by the proteasome. It is likely that artemisinin-induced degradation of residual misfolded N-CoR was triggered by the reactivation of cellular proteasomal activity originally inhibited by misfolded NCoR/PML-RAR proteins. As N-CoR was also found to be heavily ubiquitinated in NB4 cells [50], the investigation of proteasomal activities in GC011-treated NB4 cells was carried out. We observed that GC011 enhanced the degradation of the proteasome sensor expressed in 293T cells. However, MG132 treatment also restored the levels of NCoR degraded by GC011, suggesting that GC011 mediated the degradation of N-CoR through reactivating the proteasome pathway. On the contrary, MG132 treatment 67 ! failed to restore PML-RARα which was also degraded after GC011 treatment. This finding could indicate that PML-RARα could be degraded before MG132 was administered or proteasome degradation could not be the main degradation pathway. We hypothesize that GC011 may possibly enhance proteasome degradation via two modes of action: 1) directly enhancing proteasome activity, or 2) indirectly enhancing degradation through modification of N-CoR that renders it more susceptible to degradation. Proteasome activity can be regulated by proteins that bind the 19S regulatory particle (RP) and deliver ubiquitin-conjugated proteins to the proteasome, proteins that open the axial channel into the 20S core particle (CP), and ubiquitin ligases or deubiquitinating enzymes which modify proteasome-bound ubiquitin chains [165]. However, drug molecules which activate or enhance proteasome activity are rare and not well studied. PA28, PA200 and PA700 are three known types of cellular proteasome activators [166]. PA28 and PA200 bind the 20S CP to enforce channel gating in an ATP-independent manner to regulate degradation of non-ubiquitinated peptides [167, 168]. PA28 has been shown to enhance proteasome activity and improve the survival of Huntington’s disease model cells, where there is accumulation of intracellular ubiquitin-positive nuclear inclusion bodies of mutated huntingtin [169]. PA700, the 19S RP, also binds the 20S CP to induce an open pore. But unlike PA28 and PA200, PA700 regulates the degradation of ubiquitinated proteins in an ATPdependent manner [168]. Other reports suggest that oleuropein, a plant extract from Olea europea, fatty acids and SDS enhance proteasome activity by altering the conformation of the 20s CP to favor the open state [170, 171]. Presently, there has been no report on the involvement of artemisinin and direct proteasome activation which may possibly be examined in future. 68 ! Another possibility is that GC011 modifies N-CoR protein and renders it more susceptible to proteasome degradation. Proteins are tagged for proteasome degradation by post-translational modifications such as ubiquitination and posphorylation. Artemisinin derivatives have been demonstrated to enhance protein degradation through these modifications. Dihydroartemisinin accelerated the degradation of c-myc oncoprotein in tumour cells by enhancing the GSK 3β-mediated phosphorylation of c-myc [172]. In another report, artemisinin has been suggested to induce the MDM2-induced ubiquitination and proteasomal degradation of the androgen receptor protein in prostate cancer cells [77]. Proteasome activity has also been suggested to be enhanced by the inhibition of Usp14, a proteasome-associated deubiquitinating enzyme that can inhibit the degradation of ubiquitin-protein conjugates [165]. However, as N-CoR is already heavily ubiquitinated in NB4 cells, there may be a threshold to the effects of artemisinin in enhancing its ubiquitination. Hence, activating the proteasome activity directly may be a more possible mechanism. However, more work is needed to elucidate the mechanism of GC011 on regulating proteasome degradation. 4.4 GC011 inhibits autophagy in NB4 cells It was shown in this study that GC011 inhibits autophagy in NB4 cells. In another study, Gozuacik et al. reported that autophagy may be linked to cell death [104]. Since apoptosis is activated after treatment with GC011, it may be expected that autophagy will be activated. However, there are two arms of autophagy: cytoprotective and cytotoxic [173]. As we have shown, autophagy is constitutively activated in NB4 cells. It is hypothesized that cytoprotective autophagy is activated in these cells. Accumulation of misfolded proteins like N-CoR and PML-RARα may 69 ! cause the UPS to be overwhelmed as it is unable to cope with the increased load [174]. Aggregated proteins may also clog the narrow barrels of the proteasomes and inhibit proteasome activity [175]. Autophagy has been suggested as an alternative mechanism to remove these aggregated proteins. Previous studies have shown that specific ubiquitin chains associated with protein aggregates recruit p62, which in turn binds to LC3-like molecules on autophagosomes to stimulate autophagy [175, 176]. N-CoR is misfolded and insoluble only in APL cells and not non-APL cells [142]. We observed that only NB4 APL cells were able to continue growing in starvationinduced conditions. In addition, we have shown that the inhibition of autophagy reduces the intracellular ATP levels. Hence, we hypothesize that APL cells may utilize cytoprotective autophagy for the degradation of misfolded proteins to provide an alternative source of nutrients to maintain cellular growth. Thus, autophagic degradation of misfolded N-CoR not only abrogates the ER stress caused by accumulation of misfolded proteins but also confers a survival advantage to APL cells. 4.5 GC011 inhibits the PI3K/Akt pathway in NB4 cells. We have found that GC011 inhibits mTOR and Akt phosphorylation in NB4 cells. Although inhibition of mTOR is usually associated with the activation of autophagy [177, 178], we observed in this study that inhibition of mTOR and inhibition of autophagy occured after GC011 treatment. However, in situations where autophagy was activated, cell death also increased. It is possible that in these situations, it may be the cytotoxic arm of autophagy that is activated to induce cell death. However in NB4 cells, it is likely that autophagy plays a cytoprotective role suggesting that GC011 can possibly exert its effect by down-regulating cytoprotective autophagy to induce apoptosis. Apart from regulating autophagy, the Akt pathway 70 ! also regulates many other cellular processes such as cell growth, proliferation and survival [179]. Activation of the Akt pathway allows cancer cells to survive by inhibiting the proapoptotic signals and inducing survival signals, which may contribute to the malignant transformation of cells [180]. Thus, it is expected that GC011 will downregulate the Akt pathway to inhibit growth of APL cells. Cui et al. reported that the inhibition of PI3K, a protein upstream of Akt, significantly decreased autophagy levels but increased apoptosis levels in HeLa cells [181]. This was similar to what was seen in NB4 cells in this study where treatment with GC011 resulted in upregulation of apoptosis and downregulation of autophagy and the PI3K/Akt pathway. Akt is also known to elevate intracellular ATP levels via an increase in glycolysis and oxidative phosphorylation [182]. This is consistent with our observation that GC011 treatment results in a decrease in Akt activation and intracellular ATP levels along with N-CoR loss. 4.6 Hypothesis model for the action of GC011 Collectively, we hypothesize that accumulation of misfolded N-CoR and PML-RARα may inhibit proteasomal activity, which leads to the activation of autophagy. In NB4 cells, our results indicate that autophagy serves a cytoprotective function. First, it degrades the misfolded proteins to alleviate ER stress. Second, autophagy may break down the misfolded proteins to provide an alternative energy source for NB4 cell survival. We also hypothesize that GC011 induces the proteasomal degradation of misfolded N-CoR, possibly by either stimulating the proteasome activity directly or indirectly increasing degradation by acting on the posttranslational modification of N-CoR that renders it more susceptible for degradation. 71 ! Degradation of misfolded N-CoR leads to a decrease in intracellular ATP levels and exerts an anti-proliferation effect possibly because the extra energy source generated by the catabolism of misfolded N-CoR through autophagy to maintain the high requirements of tumour cell survival has now been removed. A model of the hypothesized action of GC011 is illustrated in Figure 4.1. 72 ! A B GC011! GC011! Figure 4.1. Schematic model of hypothesis in APL cells. (A) Proposed mechanism N-CoR degradation in APL cells. (B) Proposed mechanism of GC011 action in APL cells. 73 ! 4.7 Future work It has been established from previous studies that the accumulation of PML- RARα and misfolded N-CoR contribute to the pathogenesis of APL. In this study, we observe that the synthetic artemisinin GC011 inhibits the proliferation of APL cells and is capable of enhancing the degradation of misfolded N-CoR possibly through increasing proteasome activity. However, the exact mechanism by which GC011 stimulates this proteasome activity remains to be elucidated. It is crucial to determine the mechanism by which this drug exerts its effects. GC011 is an attractive potential therapeutic agent, possessing desirable properties which includes low toxicity and exerts its effects via mechanisms which are different from existing clinical drugs. Hence the next step would be to test the effects of this drug in APL mice models. APL mice can be treated with GC011 to study if the drug can result in remission of disease. 74 ! 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Genes Dev., 2001. 15(11): p. 1506-1418. [...]... environment containing high amounts of adenosine triphosphate (ATP) and Ca2+ for its proper function [85] Chaperones are key components that regulate folding of proteins They assist in the folding of newly translated proteins, refolding of misfolded proteins, prevent aggregation and facilitate proteolytic degradation [88] In the ER, three classes of proteins mediate the folding of nascent proteins – foldases,... phosphorylationdependent interaction between N- CoR and PML-RARα and subsequently dissociating N- CoR from PML-RARα This allows N- CoR to exert its effect on the differentiation of APL cells [143] On the contrary, curcumin exers its effects by promoting the accumulation of misfolded N- CoR through inhibition of the protease-mediated degradation and ERAD The net effect is the induction of UPR -induced apoptosis of APL. .. deregulated in many cancers, contributing to cancer progression and resistance to therapy PI3K is activated by growth factor receptor tyrosine kinases and G proteincoupled receptors, and catalyzes the production of PIP3 at the cell membrane PIP3 in turn recruits and activates a wide range of downstream targets, including the serinethreonine kinase Akt 3-phosphoinositide dependent protein kinase-1 (PDK1) binds... resulting in gene repression that blocks differentiation and allow uncontrolled growth of hematopoietic cells [45] In Huntington’s disease, N- CoR is localized with mSin3 exclusively in the cytoplasm of the cortex and caudate, while in the normal brain, both proteins are localized in both nucleus and cytoplasm This suggests that relocalization of N- CoR results in alteration of transcription and pathogenesis... shock protein (Hsp) 70 chaperone family It consists of an N- terminal ATPase and a C-terminal substrate binding domain Conformational changes in GRP78 regulate its binding affinity for peptides in an ATP-dependent manner The ATP-bound state allows for peptide binding, which is stabilized when ATP is hydrolyzed to ADP PDI then promotes disulphide reduction and rearrangement until proper folding is achieved... pathogenesis of disease [46] Recently, N- CoR has also found to be involved in glioblastoma multiforme (GBM) Further, increased nuclear N- CoR 8 ! expression has been found in severe grades of astrocytomas, where it maintains tumour cells in an undifferentiated state [47] 1.2.3 Current knowledge of the role of PML-RARα and N- CoR in APL pathogenesis As mentioned earlier, N- CoR is involved in the regulation of multiple... chaperones and ERAD fail to neutralize the toxicity of misfolded proteins 22 ! [20] A schematic diagram of the reglation of ER stress in APL cells is shown in Figure 1.4 [20] Further, Ng and colleagues also found genistein and curcumin to exert potent anti-proliferative effects in many APL- derived cells Genistein has been demonstrated to inhibit N- CoR misfolding in APL cells, possibly by inhibiting the... Institute in USA analysed 55 human cancer cell lines and showed that artesunate has strong anti-cancer activity against many cancer cell lines like leukemia, colon cancer, melanomas, breast, ovarian, prostate, central nervous system and renal cancer cell lines [82] Another artemisinin derivative, dihydroartemisinin, has also been shown to inhibit the growth of human ovarian cancer cells and sensitise... domains of N- CoR Repression domains (RI, RII, RIII) and SANT domains (A and B) are indicated, as are interaction domains for HDACs, nuclear receptors (I and II) and other transcription factors [36] N- CoR and SMRT can form complexes with many proteins These proteins that were consistently found in a complex with N- CoR/ SMRT include HDAC3, transducin β-like 1 (TBL1), the TBL1-related protein (TBLR1) and... formation of reactive oxygen species (ROS) [64], which causes multimerization of PML, targeting to nuclear bodies and PML sumoylation by ubiquitin-conjugating enzyme 9 (UBC9) [65] Second, ATO can also bind PML cysteines directly [65, 66], enhancing UBC9 binding to the PML RING finger and ultimately PML sumoylation [66] PML sumoylation results in the recruitment of the SUMO-dependent ubiquitin ligase and RING .. .INHIBITION OF MISFOLDED N- COR INDUCED SURVIVAL PATHWAY IN APL BY ARTEMISININ YEO HUI LING ANGIE (B.Sc.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MEDICINE... protein (Hsp) 70 chaperone family It consists of an N- terminal ATPase and a C-terminal substrate binding domain Conformational changes in GRP78 regulate its binding affinity for peptides in an ATP-dependent... inhibit N- CoR misfolding in APL cells, possibly by inhibiting the phosphorylationdependent interaction between N- CoR and PML-RARα and subsequently dissociating N- CoR from PML-RARα This allows N- CoR