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HUMAN NEUROBLASTOMA CELLS RAPIDLY ENTER CELL CYCLE ARREST AND APOPTOSIS FOLLOWING EXPOSURE TO C-28 DERIVATIVES OF THE SYNTHETIC TRITERPENOID CDDO by JENNIFER LOUISE ALABRAN Submitted in partial fulfillment of the requirements for the degree of Master of Science Thesis Adviser: Dr. John J. Letterio Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUTE STUDIES We hereby approve the thesis/dissertation of _______________________Jennifer L. Alabran_____________________ Candidate for the _____________MASTER’s________________degree.* (signed) ______Dr. James Anderson______________________________ (Chair of Committee) ______Dr. John Letterio_________________________________ ______Dr. Nicholas Ziats________________________________ (date) ___________November 6th, 2008____________________________ *We also certify that written approval has been obtained for any proprietary material contained within. ii Table of Contents: List of Tables………………………………………….………………….….…..….v List of Figures…………………………………………………………………...….vi Acknowledgements……………………………………….….............................….vii List of Abbreviations………………………………………………..…………….viii Abstract …………………………………………………………...………..……....ix Thesis Summary……………………………………………………...……………...1 a. Introduction to Pediatric Cancers…………………………...……………..2 i. Neuroblastoma………………………………...…………………..3 1. Pathogenesis and Cytogenetics…………………...…..…...5 2. Pathology ………………………………………....…..…..6 3. Treatment…………………………………………...……..7 b. Introduction to Triterpenoids…………………………………...………....8 i. Anti-inflammatory…………………………………...……………9 ii. Cytoprotection……………………………………………………10 iii. Differentiation……………………………………………………12 iv. Growth inhibition……………………………………….………..13 v. Apoptosis…………………………………………………...…....13 vi. Molecular Targets……………………………………...………...15 c. Experimental Methods…………………….…………………..….…...…17 d. Results………………………………………………………..……….….21 e. Discussion…………………………………………….……….….……...32 f. Future Directions and Conclusion……………………….……….…..….35 iii References…………………………………………………………………...….….38 iv List of Tables: Table 1: IC50 (nM) of CDDO and its derivatives in pediatric solid tumor cell lines……36 v List of Figures: Figure 1: Chemical structure of the triterpenoid oleanolic acid and synthetic oleanane triterpenoids………………………………………………………..…..22 Figure 2: Nrf2/Keap1 cytoprotective pathway………………………………………….23 Figure 3: Intrinsic and Extrinsic pathways of apoptosis…………………………….…..26 Figure 4: CDDO derivatives suppress the colony forming abilities of neuroblastoma and inhibit DNA synthesis as determined by thymidine incorporation assay……………………….………….…….…………………….34 Figure 5: CDDO derivatives deplete the S-phase in SK-N-AS neuroblastoma cells…...37 Figure 6: CDDO-Me induces apoptotic morphological changes………………….…….38 Figure 7: Exposure to CDDO-Me results in Bax confirmation change and activation in SK-N-AS and 15N neuroblastoma cells…………………………...41 Figure 8: Activation of caspases-3 and -8 in SK-N-AS neuroblastoma cells following treatment with CDDO-Me……….…….….…….……..………….…..42 Figure 9: Oral administration of CDDO-Me suppresses the growth of neuroblastoma xenografts in vivo………………………..………………………43 vi Acknowledgements: Dr. Letterio: For the opportunity that has allowed all of this to happen and your guidance over the past years. Kostya: For all of your assistance in the lab and with this project. I have learned a lot from your quiet presence. Tej: For continued support and encouragement in the lab and in life. Many thanks for all of your mentoring; taking the time to be there is greatly appreciated. Byung-gyu: For all of your help over the years especially with FACS. I wouldn’t have been able to do it without you. Dr. Khan and Adam Cheuk: For all of their input and assistance with this project. Dr. Anderson: For your understanding and guidance over the past year in helping me figure out my situation. It’s greatly appreciated. Dr. Ziats: For taking the time to be on my thesis committee. Neelima: For always being there for me and lending an ear to listen. I will miss our tea breaks. Tonibelle: For keeping me sane and providing snacks and Bahamaritas for long days, late nights and stressful days. Dr. Wurst: For continued mentoring throughout my career. Letterio Lab: For putting up with me and offering your guidance, input and assistance through the years. Family: For always believing in me and trusting my decisions whether you agree with them or not. You continue to give me the strength, determination and encouragement to accomplish my goals both in education and life, and for that I am forever grateful. I love you. Kai: For getting me through the headaches and making me realize that there is more to life than once perceived. Thanks for being there buddy. vii List of Abbreviations: CDDO: 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid CDDO-DE: CDDO-28-diethylamide CDDO-EA: CDDO-28-ethylamide CDDO-Im: CDDO-28-imidazolamide CDDO-Me: CDDO-28-methyl ester CDDO-TFEA: CDDO-28-trifluoroethylamide IC50: Half-maximal inhibitory concentration OA: Oleanolic acid RT-CES: Real Time Cell Electronic Sensing System SNS: Systemic nervous system UA: Ursolic acid viii Human neuroblastoma cells rapidly enter cell cycle arrest and apoptosis following exposure to C-28 derivatives of the synthetic triterpenoid CDDO Abstract By Jennifer Louise Alabran Synthetic triterpenoids, such as 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) and its derivatives, belong to an extremely potent class of new anti-cancer therapeutic agents characterized by high anti-tumor potency and low toxicity to normal tissue. This work is the first to investigate the effects of C-28 derivatives of CDDO in several pediatric solid tumor cell lines in vitro and in a mouse xenograft model of human neuroblastoma. Using C-28 CDDO analogs we determined an IC50 in the range of 5 – 630 nM depending on the method used. We show partial G2/M arrest, conformational activation and mitochondrial translocation of Bax protein, as well as activation of caspases -3 and -8 following treatment with CDDO-Me. Tumor xenograft experiments in mice demonstrated a significant delay in tumor growth in mice fed a diet containing CDDO-Me. Overall, CDDO analogs appear to be promising for development as novel therapeutic agents for these high-risk pediatric solid tumors. ix Thesis Summary Neuroblastoma is the most common extra-cranial solid tumor of childhood and accounts for 7-10% of all pediatric cancers 1. Advances in early detection and surgery have largely been responsible for improved long-term survival rates, which plateau at 60% at 5 years 2 . However, the disease is metastatic at diagnosis in more than 50% of cases with 85% of the tumors high grade. The outlook for these high-risk patients remains poor and prompts the development of novel therapeutic drugs to treat this disease. Currently, there is a great emphasis on the development of molecularly targeted therapies for cancer with abundant evidence that multiple signaling pathways, including NF-κB 3, Jak/STAT 4, cyclooxygenase (COX) 5, p53 6, p38-MAPK 7 and others are active and linked directly to the viability, progression and chemotherapy resistance of neuroblastoma. These pathways work in interconnected networks suggesting that multifunctional agents that are capable of targeting multiple pathways, rather than specific molecules, would be a more effective strategy to inhibit growth and induce cell death in neuroblastoma. Synthetic triterpenoids have emerged recently as a potent class of small molecules that are highly effective in both the prevention and treatment of cancer 8. An oleanane triterpenoid, 2-cyano-3, -12-dioxoolean-1, 9-dien-28-oic acid (CDDO) and its derivatives were originally developed as inhibitors of nitric oxide production in macrophages and as anti-proliferative agents for tumor cells 9-11. It is now clear that the synthetic triterpenoids are multifunctional, having the ability to modulate the function and expression of many relevant targets involved in signaling pathways and modulation of genes linked to oxidative stress and cell survival. The efficacy of the triterpenoids has been 1 demonstrated in preclinical studies that include models of prevention or treatment in various types of cancer including leukemias, melanoma, breast, ovarian, prostate, pancreatic, colon, non-small cell lung, and multiple myeloma10, 12-18. The aim of this thesis is to examine the activity of the synthetic triterpenoids in the spectrum of common pediatric solid tumors. Here we report for the first time the potency and mechanisms of the activity of several of the triterpenoids in neuroblastoma as well as other pediatric solid tumors including rhabdomyosarcoma, Ewing’s sarcoma, and osteosarcoma. At nanomolar concentrations, CDDO and its derivatives were able to induce apoptosis and cell cycle arrest in the cell lines tested. This data supports the development of synthetic triterpenoids for clinical evaluation as a new and needed approach for the treatment of these common forms of childhood malignancy. Introduction to Pediatric Cancer: Pediatric cancers result in approximately 1% of all new cancer cases in the U.S. annually. Although the five year survival rates have greatly increased over the past 25 years, malignant neoplasms remain to be the second leading cause of death among children 19. Pediatric cancers are remarkably different than adult cancers in both prognosis and histology. When compared to adult malignancies, very few pediatric cancers can be linked to environmental factors suggesting that genetics may play a very key role in development of these tumors. Pediatric cancers most commonly peak at two different time points in a child’s life, either in the first few years where neuroblastomas, Wilms tumor, retinoblastoma, rhabdomyosarcoma and medulloblastomas are common, or in adolescence where bone malignancies and leukemias are prominent. 2 Treatment of childhood malignancies is very complex since it depends on the correct diagnosis, accurate staging, and identification of the subtype. The best chance of a child going into and remaining in remission is during the initial course of treatment so multidisciplinary and multimodal therapy is used. Current chemotherapies are almost always used in combination with other chemotherapies resulting in a mix of alkylating agents, antimetabolies, antibodies, hormones and/or toposomerase inhibitors. The increased metabolic and cell cycle activity of malignant cells makes them more susceptible to the cytotoxic effects of chemotherapy. The problem remains that the chemotherapy also exerts damage to normal cells leading to acute or late adverse effects in the patient. Acute adverse effects are nearly always reversible and include myelosuppression, immunosuppression, nausea, vomiting and alopecia. Late adverse effects can occur many years after treatment and usually never reversible. These include subsequent malignancy, early mortality, infertility, long term organ damage, reduced stature, cardiomyopathy, osteoporosis, neurocognitive impairment, mood disorders and depression. New therapeutic approaches need to focus on the use of drugs in combination with chemotherapy that may alleviate or eradicate these side effects. Neuroblastoma Neuroblastoma is defined as an embryonal malignancy of the systemic nervous system (SNS) derived from primordial neural crest cells that during fetal development are destined for the adrenal medulla and the SNS 20. Within this category, ganglioneuroblastomas and ganglioneuromas are also included. The neural crest is a transient component of the ectoderm of an embryo during neural tube formation. It gives 3 rise to neurons and glia of the autonomic nervous system, medulla cells of the adrenal glands, pigment containing cells of the epidermis, and skeletal and connective tissue. Although neuroblastoma can develop at any site of the SNS tissue, most cases occur in the abdomen either in the adrenal gland or in the retroperitoneal sympathetic ganglia19. Neuroblastomas most commonly arise in the adrenal medulla which is made up of chromaffin cells serving as the body’s main source of adrenaline, norepinephrine, and dopamine. Each year 700 children less than twenty years of age are diagnosed with a neuroblastic tumor, with 97% of these cases being neuroblastoma. Neuroblastoma is the third most common childhood malignancy (7.8%) and the most common among infants where the rate is almost double leukemia, the second most common occurring in the first year of life 21. Prognosis is dependent on the age of the patient, the stage of the cancer, and the molecular biology and cytogenetic characteristics of the tumor. Most patients are asymptomatic which leads to 50-60% of neuroblastomas being metastatic at diagnosis21. Metastases are most common in the long bones, skull, bone marrow, liver, lymph nodes and skin. In infants, the five year survival rate is 83%, with a markable decrease with age where older children 1-4 years old and children >4 years old the survival rate is 55% and 40%, respectively22. Relatively little is known about the etiology of neuroblastoma but is it thought that certain exposures occurring before conception and during gestation may have a key role in the development. Maternal factors such as opiate consumption, foliate deficiency, toxic exposures, congenital abnormalities and gestation diabetes mellitus have all been noted. Although most neuroblastomas are sporadic, hereditary neuroblastomas have been seen in 1-2% of 4 the cases23 in which the disease is inherited in an autosomal dominant pattern with incomplete penetrance and broad clinical behavior24. Inherited cases of neuroblastoma usually occur earlier in life than sporadic cases (9 vs. 17 months). Clinical and molecular risk factors include amplification of MYCN, loss of heterozygosity (LOH) of chromosome 1, and hyperdiploidy, all of which will be discussed in detail below. Pathogenesis: Adrenal neuroblastoma develops from residual microscopic neuroblastic nodules that can be detected in the fetus between the fifteenth and twentieth weeks and regress by the time of birth or shortly after. The transformation of cells may result from failure to respond fully to normal signals that stimulate morphologic differentiation leading to malignancy. Loss of heterozygosity is found in 50% of the cases, with chromosome 1p, 11q and 14p frequently deleted. Deletion of 1p is the most common alteration and is associated with poor prognosis 25, 26. Part of this is due to the amplification and overexpression of MYCN which is also associated with 1p deletions 27. Deletions in 11q and 14p are found in 25-50% of the cases and appear to have a distinct tumor subtype 28. A gain of chromosome 17q occurs in 50% of the cases which presents as an aggressive phenotype 29. In addition, an alteration in the total DNA content resulting from mitotic dysfunction is an important indicator of both outcome and response to therapy 30. Hyperdiploidy tends to have a lower tumor stage, better response to chemotherapy and overall a better prognosis. MYCN amplification is used as a prognostic factor for the diagnosis of neuroblastoma and was one of the first genetic markers used to identify proper 5 treatments. MYCN amplification occurs in approximately 25% of all neuroblastoma cases and is strongly associated with advanced stage neuroblastoma and rapid tumor progression 31, 32. MYCN is an oncogenic transcription factor that participates in the regulation of cell growth and apoptosis. Under normal conditions, cells with aberrant expression of MYCN are eliminated through apoptosis; however, cells with defects in their apoptotic machinery and deregulated MYCN may lead to tumorgenesis. It has been shown that amplification of MYCN accelerates cell cycle progression in neuroblastoma cells 33. This amplification has long been known that it leads to overexpression at both the protein and RNA levels 34, 35. RNAi has been used to show MYCN role in neuroblastoma tumorigenesis 36 and there are reports that silencing of MYCN by siRNA results in increased apoptosis and differentiation 37. Pathology: Neuroblastomas are classified by the balance between neural-type cells (primitive neuroblasts, maturing blasts, or gangalion cells) and Schwann-type cells (blast or mature) leading to three types of cancer: neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Neuroblastoma is the most undifferentiated appearing and the most aggressive of the three types. Within itself, neuroblastoma can be broken down into three categories: undifferentiated, poorly differentiated, or differentiated. Along with the stromal component, this can be used to predict the outcome of the patient. Neuroblastoma is composed of almost entirely of neuroblasts with very few stromal cells. Under the light microscope, neuroblastoma cells appear as a monotonous collection of small, round, blue cells having a similar morphology to osteosarcomas, 6 Ewing’s sarcomas, and rhabdomyosarcomas. Due to this, either electron microscopy is used or the cells are stained with a specific monoclonal antibody that recognizes neural tissue to identify it as neuroblastoma. Differentiated neuroblastomas have hyperchromatic nuclei and inadequate cytoplasm and can form Homer-Wright rosettes. Ganglioneuroblastomas are intermixed stroma-rich with an increased number of Schwannian cells. The neuroblasts are clustered together in foci or nests surrounded by the Schwann cells. Ganglioneuroblastomas usually have an intermediate malignant potential. Ganglioneuromas are Schwann cell dominated with maturing or fully mature ganglion cells present throughout. This is usually seen in older children (5-7 years of age) rather than the more aggressive neuroblastomas, and is mostly consider benign although the tumor can metastasize. Ganglioneuromas have an excellent prognosis even when the complete removal of the tumor is not possible. Treatment: What makes neuroblastoma so difficult to study and treat is that there is a broad spectrum of clinical behavior ranging from spontaneous regression to maturation to benign or aggressive disease with metastases leading to death38. The primary factor that determines the outcome of the patient is the degree of metastatic spread at diagnosis. Spread to the lymph nodes adjacent to the primary tumor does not affect the outcome as much as distant metastases. A standard system for staging has been defined based on the resectability and spread of disease to distant sites where age plays some role as the older the child, the less favorable the outcome. Children less than one year of age commonly have localized tumors and better survival rate than older children. 7 Surgery is the primary treatment for those patients who fall into the low-risk category. How much of the primary tumor that can be removed is determined by the tumor location, mobility, its relationship to major nerves and blood vessels, the presence, if any, of distant metastases, and age. Prenatal diagnosis can be performed detecting small adrenal masses by ultrasound; however, treatment (surgery) is often delayed in infants due to a high rate of spontaneous regression after birth. Common chemotherapies include cyclophophamide, carboplatin, cisplatin, etoposide, and adriamycin where patients who have a more aggressive tumor may also receive vincristine and doxorubicin. Specific adverse effects for neuroblastoma patients include vascular injury at the time of surgery which can lead to ischemic loss of the kidney and endocrine effects such as thyroid dysfunction and infertility in addition to the effects already mentioned. The majority of neuroblastomas are initially chemoresponsive with approximately 80% responding during induction therapy; however, more than half of these will progress to therapy-resistant disease22. A better understanding of the tumor-specific evasion of apoptosis may be the key to understanding the mechanism of resistance. Currently there is an active research interest in pediatric oncology for drugs that generalize targeting of tumor cells with drugs that induce apoptosis or target angiogenesis. It has been thought that the combination of deregulated oncogenic signaling, like the amplification of MYCN and abrogated apoptotic signals, may lead to progression and resistance. Introduction to Triterpenoids Triterpenoids have been used in Asian countries for many years for medicinal purposes. There are more than 20,000 natural triterpenoids which are biosynthesized in 8 plants by the cyclization of squalene 39. Two of these triterpenoids, oleanolic acid (OA) and ursolic acid (UA), have been shown to have weak anti-inflammatory and anittumorigenic properties 40, 41. This work will focus on the synthetic oleanane triterpenoids which are effective in prevention and treatment of various cancers 8, 12, 15, 42-44. In order to increase the anti-inflammatory and anti-tumorigenic properties, modifications have been made to the basic OA and UA structures through medicinal chemistry at Dartmouth University in Dr. Sporn’s laboratory. They have tested the new synthetically derived compounds, such as 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) and its derivatives (Figure 3), and evaluated them for their anti-inflammatory properties 10. CDDO’s biological applications vary dose dependently where CDDO can suppress inflammation, activate cytoprotective pathways, induce differentiation, inhibit proliferation, and induce apoptosis in vitro. Each of these properties will be discussed. Anti-inflammatory Many cancers arise from sites of infection, chronic irritation and inflammation. Recently inflammation has been identified is a critical component of tumor progression where the microenvironment participates in malignant growth promoting proliferation, survival and migration 45. It is known that the overexpression of iNOS and COX2 can drive carcinogenesis 45, 46. CDDO can suppress the induction of both of these in stimulated primary macrophages and has been shown to block the de novo synthesis of COX2 in stimulated colon myofibroblasts 10. CDDO concentration to inhibit de novo synthesis of iNOS has been reported at an IC50 of 0.4 nM11. 9 Cytoprotection The significant components of CDDO are the α, β-unsaturated carbonyl groups located at key positions on rings A and C of oleanolic acid which are essential for the anti-inflammatory potency 47(Figure 1). These functional groups allow for the formation of Michael additions with nucleophilic targets such as Keap1, which has multiple cysteine residues, activating Phase 2 responses 48. Carbons 1 and 9 then act as Michael acceptors and are targeted by sulphhydryl groups. This reaction does not react indiscriminately with all cysteine residues within a protein as the redox potential of the cell and the accessibility of cysteine residues within the protein structure are important factors. Potentially CDDO could interact with other nucleophilic groups on lysine, arginine, or histidine on targeted proteins or alter post-translational modifications such as acetylation or phosphoraylation but no data to support such mechanisms has yet to be defined. 10 OA CDDO-Im CDDO CDDO-EA CDDO-Me CDDO-DE CDDO-TFEA Figure 1: Chemical structure of the triterpenoid oleanolic acid and synthetic oleanane triterpenoids: OA, Oleanolic acid; CDDO, 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid; CDDO-Me, CDDO-28-methyl ester; CDDO-Im, CDDO-28-imidazolamide; CDDO-EA, CDDO-28ethylamide; and CDDO-DE, CDDO-28-diethylamide; CDDO-TFEA, CDDO-28-trifluoroethylamide. This reaction activates an intrinsic mechanism used by cells to deactivated oxidative stress 49, 50 referred to as the phase 2 response. Phase 2 genes encode for various cytoprotective proteins including enzymes such as glutathione synthesis and hemeoxygenase-1 (HO-1) as well as many others. In response to oxidative stress, Nrf2, a transcription factor, is released from its inhibitor protein, Keap1, activating an antioxidant response element (ARE) on the promoter of the phase 2 response genes 49, 51, 52(Figure 2). It is important to note that although Nrf2 can activate some phase 1 response genes, CDDO is selective and can only induce phase 2 enzymes 47. One major effect of this response is the reduction of reactive oxygen species (ROS); however, at the same time 11 CDDO is bifunctional and at higher doses it can actually increase oxidative stress and cause apoptosis. It is important then to consider the dose and the expected outcome. This phenomenon may be due to CDDO’s affinity to different targets where a low concentration might interact with Keap1 activating cytoprotective pathways and higher concentrations may interact with proteins that have lower affinities such as IKK and induces apoptosis. Figure 2. Nrf2/Keap1 cytoprotective pathway. Activation of this pathway leads to the upregulation of phase 2 response genes. In the absence of cellular stress, Nrf2 is bound by Keap1 and Nrf2 is directed for proteasomal degradation. The triterpenoid forms a Michael Addition with the Keap1/Nrf2 complex releasing Nrf2 and allows it to accumulate within the nucleus and activate cytoprotective genes. Differentiation Abnormal differentiation is a hallmark of many types of cancer. At concentrations higher (IC50 = 100-300 nM) than those required to inhibit de novo synthesis of iNOS, CDDO is a potent inducer of differentiation pathways in malignant cells. CDDO-Me and CDDO-Im have been shown to induce differentiation in monocytic leukemia cells at doses much lower than the parent compound, CDDO 12, 53. 12 Growth Inhibition CDDO can inhibit proliferation (IC50 = 0.02 – 2 μM) but at higher doses than those required for the anti-inflammatory properties. CDDO and its derivatives have been shown to inhibit proliferation in various malignant cells at nanomolar concentrations 54, 55 . CDDO is also able to regulate the expression of several key cell cycle proteins such as p21 and MYC 56, 57. Highly aggressive forms of neuroblastoma usually over express MYC so CDDO may be a potential inhibitor of this overexpression and advanced tumor aggression. Apoptosis Apoptosis is a form of programmed cell death that involves a series of biochemical events that lead to the death of a cell without an inflammatory response. Various morphological changes occur during this process such as a loss of membrane asymmetry and attachment, cell shrinkage, membrane blebbing, nuclear fragmentation and chromatin condensation. Apoptosis is regulated by a family of cysteine proteases, the caspases, which are signaling mediators that control a complex web of downstream events that lead to cellular death. Other key players in apoptosis are members of the Bcl-2 family that regulate apoptosis by controlling mitochondria permeability. Antiapoptotic members such as Bcl2 reside in the outer wall of the mitochondria inhibiting the release of cytochrome c. Proapoptotic members such as Bid and Bax are located in the cytosol but translocate to the mitochondria when apoptosis is signaled leading the release of cytochrome c in response to the death stimuli. Cytochrome c then binds Apaf-1 and forms an apoptosome 13 with procaspase-9. The activation of caspase-9 leads to cell death via activating caspase3. Cells that are apoptotic display phosphatidylserine on their cell surface marking the cell for phagocytosis. Apoptosis is initiated most commonly through the intrinsic or extrinsic pathway (Figure 3). The intrinsic pathway occurs in response to stress signals such as DNA damage and hypoxia and is mitochondria-dependent. Through this pathway the activation of proapoptotic proteins causes the release of cytochrome c and Smac/DIABLO from the mitochondria and the activation of caspase-9 through Apaf-1 leading to cell death. The extrinsic pathway is mediated by death receptors and is initiated one way by the interaction of Fas and FasL. This results in oligomerization of the receptors and recruitment of adaptor proteins, such as FADD and caspase-8. Activated caspase-8 directly activates other caspases and triggers apoptosis. Figure 3. Pathways of apoptosis. Cellular death can occur through either the extrinsic or intrinsic pathway once the cell is exposed to the triterpenoid. 14 CDDO can induce apoptosis by either of these pathways 13, 58, 59. The initiation to the apoptotic cascade is different and unknown for each type of cancer. It is known that at higher concentrations, CDDO disrupts the redox balance of the cell decreasing intracellular and mitochondrial glutathione levels and therefore reducing the antioxidant capacity of the cell. CDDO has been shown to induce apoptosis in leukemia patients, even when these cells are resistant to standard chemotherapies. Tissue harvested from the same patient was used to show that CDDO is not toxic to normal lymphocytes of those patients 16, 60. This selectivity may occur due to the higher endogenous levels of oxidative stress in the malignant cells 61. CDDO can activate c-JNK and the p38 stress pathway as well as inhibit NF-κB and STAT signaling 18, 62-64. Drugs that target cancer cells and able to induce apoptosis are obviously important for treatment of pediatric malignancies and should be identified. Molecular Targets Very little is actually known on the overall mechanism of CDDO on different cellular processes. This work is focused toward delineating the mechanism of CDDO in inducing apoptosis in neuroblastoma cells. Individual proteins and pathways have been identified as direct targets of CDDO but none of these provide sufficient evidence for the full story. One challenge in solving this issue is due to the reversibility of the Michael additions between CDDO and the interacting nucleophiles 48, 65. It is thought that CDDO may trigger a cellular switch by binding to one of the targets and induces a cellular response without staying bound to the target. 15 Two known direct targets of interest are Keap1 and IKKβ. The NF-κB pathway regulates cell cycle, differentiation and apoptotic genes and is known to be constitutively active in many human cancers 65. CDDO inhibits NF-κB by directly binding and inhibiting its activator, IKKβ 64, 66. Although the mechanisms of these direct targets are well understood, they do not account for all of the biological properties of CDDO. It is also known that CDDO can mimic signaling by members of the TGFβ superfamily 67 which play an important role in the regulation of inflammation, cell growth, differentiation and apoptosis. Much is known about STATS expression in cancer and how inappropriate signaling can lead to disease. It has been shown that CDDO-Im rapidly suppresses both constitutive and IL-6 inducible STAT3 and STAT5 phosphorylation in myeloma and lung cancer cells leading to growth arrest and apoptosis 18 . There has been a strong effort recently in developing multifunctional drugs to be used for the prevention and/or treatment of cancer. Rather than having a drug that binds specifically to a kinase involved in signaling transduction, it would be beneficial to have a drug that could modulate the activity of regulatory pathways that may be dysfunctional in malignant cells and tissue. It is clear that CDDO could potentially be one of these drugs and worth continued exploration to identify mechanism and clinical relevance. 16 Material and Methods Synthetic Triterpenoids. CDDO, CDDO-Me, CDDO-Im, and other amide derivatives were synthesized as described 9, 11, 13. Stock solutions (10 mM) were made in DMSO and frozen until used. Cell Culture. The following cell lines were used: human umbilical vein endothelial cell line, HUVEC; rhabdomyosarcoma, RD and RH41; osteosarcoma, SA-OS and U2-OS; Ewing’s sarcoma, RD-ES, TC32, TC106 and TC71; neuroblastoma, CHP-134, IMR5, NB1691, IMR32, LAN1, NB-EB, SK-N-AS, SY5Y, SK-N-SH, SK-N-BE2, SK-N-FI, 15N and SK-N-DZ, were grown 37ºC, 5% CO2 in 1x RPMI 1640 with L-glutamine (Invitrogen, Carlsbad, CA) with 100 units/ml penicillin, 100µg/ml streptomycin, and 10% FBS (Gemini Bio-Products, West Sacramento, CA). 3 H-thymidine incorporation assay. Cells were plated in triplicates at 1 x 105 cells per well in 96-well plates and cultured in 100µl of medium containing CDDO-Me (0-250nM) overnight. After 22 hours, cells were pulsed for 2 hours with 3H-thymidine (Perkin Elmer, Wellesley, MA) at 1µCi/25µl of medium per well. Samples were harvested onto a filter with the MicroBeta FilterMate (Perkin Elmer), prepared with Betaplate Scint (Perkin Elmer) and read with the Wallac MicroBeta Tri-Lux (Perkin Elmer). Colony Forming Assay. Cells were plated in triplicate in 60 mm dishes at 4x103 cells/dish with serial dilutions of triterpenoid (0 – 200nM) and incubated for least 7 days until colonies of 50-100 cells/colony were formed. Medium was removed, and colonies 17 were stained with 1% Crystal Violet in 10% ethanol and rinsed twice with water. Colonies were counted using Quantity One Software (BioRad, Hercules, CA). RT-CES assay. Continuous cell proliferation was measured by RT-CES (ACEA Biosciences Inc., San Diego, CA). Briefly, cells were seeded at 104 cells/well on 16-well e-plates (ACEA) in 200µl of medium. 20 hours later, 20 µl of medium containing either CDDO-Me from 0.13 µM to 1 µM or DMSO was added. Proliferation was monitored every hour. Dose response curves were plotted using the ACEA RT-CES SP software (ACEA), and IC50 were calculated for 24 hours. Analysis of apoptotic morphology. 15N and SK-N-AS cells were treated with CDDOMe (250 nM) for 10 hours, harvested and washed twice with cold PBS. Cells were then used to make cytospins and stained with Hema 3 Stain Set (Biochemical Sciences Inc., Swedesboro, NJ). Western Blot Analysis. Cells were treated with 250 nM CDDO-Me for 6 hours and then harvested using 1x RIPA buffer containing Protease Inhibitor Cocktail (Roche, Indianapolis, IN) on ice for 20 minutes. Extracts (50 μg) were resolved by Novex Trisglycine gels (4-20%; Invitrogen Corporation, Carlsbad, CA) and transferred to nitrocellulose membranes (Invitrogen), followed by Ponceau S staining and blocking by 5% non-fat milk in TBST (0.05% Tween 20, FisherBiotech, Fair Lawn, NJ). Membranes were probed with HO-1, Bax (Santa Cruz Biotechnology, Santa Cruz, CA), or Hsp70 (Cell Signaling Technology, Danvers, MA) antibodies overnight at 4°C. HRP-conjugated 18 secondary antibodies (SouthernBiotech, Birmingham, AL) were then used followed by ECL detection (Pierce). Membranes were stripped (Pierce) and re-probed using β-actin antibody (Sigma, St. Louis, MI). Immunoprecipitation of activated Bax. Protein G Sepharose beads (20 μl, Pharmacia) were pre-incubated with 2 μg of anti-Bax monoclonal antibody (6A7) overnight on a rotating disk at 4°C. SK-N-AS cells (1 x 106 cells/100 mm dish) were treated with either CDDO-Im, CDDO-Me, staurosporine (STS) or an equal amount of DMSO and harvested in CHAPS buffer after either 6 or 24 hours. Supernatants were collected (30 minutes, 14,000 rpm, benchtop centrifuge, 4ºC). Beads were washed three times with 200 µl CHAPS buffer, and cell extracts (1 mg of total protein) were added to the 6A7 antibody/beads complexes. After 2 hour incubation on a rotating disk at 4°C, beads were collected by centrifugation (1000 rpm, benchtop centrifuge, 3 seconds, 4°C), washed three times with 100 μl of CHAPS buffer, re-suspended in 40 μl of 2X Laemmli buffer, boiled for 5 minutes and resolved on SDS-PAGE alongside with the input samples (20 μg of total protein in CHAPS lysis buffer). Western blot analysis was performed using antiBax (N20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Preparation of mitochondrial and cytosolic fractions. Cells were incubated for 6 hours with 0, 10, 100, or 250 nM CDDO-Me or 1 µM of staurosporine in complete RPMI medium. Cells were then harvested and homogenized in 10 mM Tris HCl (pH 7.4) containing 0.32 M sucrose and 1 mM potassium EDTA using a Dounce homogenizer with four strokes of loosely fitting pestle. The homogenate was centrifuged at 1,000 g for 19 5 minutes to remove cell debris and nuclei. The mitochondria were collected by centrifuging the supernatant at 13,000 g for 5 minutes, with the resulting supernatant used as the source of cytosol. Both pellet and supernatant were subjected to Western blotting, as described above. Caspase activity. 15N cells were treated for 6 hours with 0-250 nM of CDDO-Me. Cells were collected by centrifugation at 600 g for 5 minutes at 4°C and then washed once with PBS. Cell pellets were re-suspended in 1x lysis buffer (250 mM HEPES, pH 7.4, 25 mM CHAPS, 25 mM DTT) at a concentration of 500 µl per 107 cells. The homogenates were centrifuged 14,000 g for 15 minute at 4ْC and 5µl cell extract was added to 5µl 1x assay buffer (20 mM HEPES, pH 7.4, 0.1% CHAPS, 5 mM DTT, 2 mM EDTA and 50% sucrose). Specific caspase inhibitors were added to the extracts and manufacturers’ procedures were carried out from the Caspase-3 or caspase-8 Assay Kit (Sigma, St. Louis, MI). AMC release was determined by measuring fluorescence with excitation at 360 nm and emission at 460 nm using Victor3 1420 Multilabel Counter (PerkinElmer, Wellesley, MA). Cell cycle analysis. SK-N-AS cells were exposed to 500 nM CDDO-Me for 24 hours, cell cycle assay was performed by BrdU flow kit (Pharmingen, San Jose, CA). Briefly, cells were pulsed with BrdU (10 µmol/L) for 2 hours. Cells were then harvested, fixed and incubated with 20 µL anti-BrdU-FITC for 20 minutes and then 10 minutes with 7AAD. 20 In vivo tumor model. Female 8-10 week old NIH-III mice (SAIC, Fredrick, MD) were housed under pathogen free environment. SK-N-AS cells were prepared by washing 2x with cold HBSS and brought to a final density of 107cells/ml in cold HBSS. A total of 5 x106 cells in 0.5 ml were injected subcutaneously into the right flank of each mouse, and they were started on a powdered diet with or without CDDO-Me (100 mg/kg of diet) on the day when tumor cells were injected. Mice were examined three times each week and all tumors were measured by caliper at least once each week. Mice were sacrificed when tumor reached a volume greater than 2500mm3 or when the mouse showed significant morbidity, and were excluded from the study if an intraperitoneal tumor was formed. Results CDDO and its derivatives inhibit the proliferation and viability of various pediatric solid tumors at nanomolar concentrations. It has been shown previously that the CDDO derivatives, CDDO-Me and CDDO-Im, have significant anti-tumor activities for various tumor types with IC50s in the nanomolar range8 and at the same time, very low, if any, side effects were detected at clinically relevant concentrations 13. Therefore, CDDO-Me will be used mainly throughout this study as it is one of the only derivatives currently in Phase I trials. Four human neuroblastoma cell lines (NB1691, 15N, LAN-1 and SK-N-AS) and one Ewing’s sarcoma (TC106) were tested for colony formation in the presence of four compounds: CDDO, CDDO-Im, CDDO-Me and CDDO-TFEA (Table 1, Figure 4A, B, D). In addition, SK-N-AS and TC106 cells were tested for colony forming in the presence of CDDO-EA and CDDO-DE (Table 1). Neuroblastoma cell lines displayed 21 significant sensitivity to CDDO-Im, CDDO-TFEA and CDDO-Me at concentrations less than 150 nM (see Table 1 for IC50 and Figure 4A, B, D for survival curves). SK-N-AS and TC106 cells were also sensitive to CDDO-EA and CDDO-DE at doses less than 200 nM. The parent compound, CDDO, had no inhibitory activity on colony formation by NB1691, LAN-1, SK-N-AS and TC106 cells when tested at concentrations less than 200 nM. However, it did inhibit colony formation by 15N cells within this concentration range. The same neuroblastoma and Ewing’s sarcoma cell lines were tested for 3Hthymidine incorporation into DNA after treatment with 0–250 nM CDDO-Me (Figure 4C). Similar to colony-formation assays, 3H-thymidine incorporation was decreased by 50% at 30-150 nM CDDO-Me and by greater than 95% at 250 nM CDDO-Me in all cell lines. At least one cell line, NB1691, showed a transient increase in thymidine incorporation at a low (4-16 nM) dose of CDDO-Me, followed by a dose-dependent decrease in 3H-thymidine incorporation. As in the colony formation assay, the 15N cell line showed the highest sensitivity to CDDO-Me with IC50 at ~30 nM. 22 Figure 4. CDDO derivatives suppress the colony forming abilities of neuroblastoma and inhibit DNA synthesis as determined by thymidine incorporation assay. A, B, and D: Colony forming assays. Experiments were performed in triplicates. Error bars represent standard errors of survival fraction for each dose of the drug. C: Thymidine incorporation assays were performed in triplicate. Error bars represent standard errors of means survival fraction for each dose of the drug. Using the RT-CES system, the effect of CDDO-Me was tested on thirteen different neuroblastoma cell lines, as well as two rhabdomyosarcoma, two osteosarcoma, and three Ewing’s sarcoma cell lines. All of the cell lines tested were sensitive to CDDO-Me in a time and dose-dependent manner. The IC50 values were determined from dose response curves generated at 24 hours by this method ranged from 160nM to 630nM for all pediatric solid tumors that were evaluated (Table 1). The principle of RT-CES is based on an impedance measurement by electrodes placed in each well with cultured cells. Impedance is proportional to the area occupied by attached cells, which increases as cells proliferate and decrease when they die and detach 23 during either late apoptosis or necrosis. The IC50 determined by the RT-CES assay indicates the concentration of the compound required to reduce the area occupied by attached cells by 50% from the time point when the drug is added (Table 2). We compared the RT-CES and the end point WST-1 assays on SK-N-AS cells treated with different concentrations of CDDO-Me (Table 1). Both methods produced similar IC50 values (480 nM for CDDO-Me in SK-N-AS cells), suggesting that a decrease in the area occupied by cell population due to death correlates with the drop in metabolic viability of the cells. However, the IC50 values for CDDO-Me determined by both the colony forming and 3H-thymidine incorporation assays were significantly (4 – 35 fold) lower than the IC50 determined by RT-CES assay in the same cell lines. This indicates that the inhibition of proliferation and DNA replication determined using colony forming and 3Hthymidine incorporation assays requires significantly lower concentrations than cell detachment from substrate that are determined by the RT-CES and WST-1 assays. 24 CDDO-Me RTCES Cell line Type HUVEC Umbilical Vein Endothelial cells 110 RD Rhabdomyosarcoma 280 RH41 SA-OS Osteosarcoma CDDOEA CDDODE Colony forming Colony forming Colony forming Colony forming Colony forming 195 125 95 35 Colony forming 3Hthymidine 117 43 >200 37 95 35 75 >200 35 85 415 Ewing's sarcoma 309 402 TC71 373 TC106 IMR32 CDDOTFEA 300 TC32 CHP134 CDDOIm 219 U2-OS RD-ES CDDO Neuroblastoma 201 232 IMR5 610 LAN1 160 LAN5 250 NB-EB 305 NB-1691 283 8 137 >200 5 125 SKNAS 418 115 76 >200 30 170 SKNSH 630 SKNBE2 273 SKNDZ 525 SKNFI 465 SY5Y 181 25 30 5 35 15N 25 Table 1: IC50 (nM) of CDDO and its derivatives in pediatric solid tumor cell lines determined by RTCES, 3H-thymidine incorpation, and/or colony forming assays. Derivatives tested were CDDO-Me, CDDO, CDDO-Im, CDDO-TFEA, CDDO-EA, and CDDO-DE. Neuroblastoma cells exposed to CDDO-Me show impaired transition through the G2/M checkpoint and depletion of the S-phase population. To determine the effect of CDDO-Me on cell cycle distribution, standard cell cycle analysis was performed on the neuroblastoma SK-N-AS cell line treated with CDDO-Me. Cells were exposed to 200 nM of CDDO-Me for 24 hours and then a BrdU incorporation assay was performed as described in Materials and Methods (Figure 5). A decrease of approximately 60 fold in S phase was found for all three compounds. Also, increases of approximately 1.5 fold in 25 sub-G1/G0, which was indicative of DNA degradation and apoptosis, was observed (Figure 5). Cells treated with CDDO-Im, the most potent of all three compounds, showed 1.5 fold decreases in the G1/G0 phase. This profile for cell cycle distribution has been observed in neuroblastoma cells when exposed to other agents that induced apoptosis, including N-(4 hydroxyphenyl) retinamide 68. 35 Population, % 30 * * 25 SubG1/G0 G1/G0 G2/M S * 20 15 10 5 0 ** ** ** e A ted O-Im O-TFE O-M rea D D t D D n C U C CDD Figure 5: CDDO derivatives deplete the S-phase in SK-N-AS neuroblastoma cells. Cell cycle profile as percentage of cells in each cell cycle phase by FACS analysis. Cells were exposed to 200 nM of CDDO-Me, CDDO-Im, CDDO-TFEA, or DMSO as a control. Experiments were performed in triplicates. Error bars represent standard errors of means for each set of data. * p<0.05, ** p < 0.01, when compared to control. Neuroblastoma cells exhibit apoptotic morphology after CDDO-Me treatment. To study morphological and molecular apoptotic changes after treatment with triterpenoids, we chose CDDO-Me, since it displayed high biological activity in RT-CES, colony forming and 3H-thymidine incorporation assays. Neuroblastoma cell lines (15N and SKN-AS) were treated with 250 nM CDDO-Me for 10 hours. After treatment, cytospin slides were prepared and stained. Microscopy analysis revealed extensive nuclear 26 fragmentation and membrane blebbing in treated 15N cells as early as 10 hours after treatment (Figure 6B). Similar membrane blebbing was detected in SK-N-AS cells, however, no significant nuclear fragmentation at the 10 hour time point was visible, although, some nuclei were picnotic and had prominent lobes in the treated cells (Figure 7D, arrows). This difference in responses to CDDO-Me in two cell lines correlates with higher sensitivity of 15N cells to CDDO-Me in comparison to SK-N-AS cells as determined by colony forming assay (Table 1, Figure 4A). A C B D Figure 6: CDDO-Me induces apoptotic morphological changes. Cells were treated with 250 nM of CDDO-Me for 10 hours. After treatment, cells were analyzed by cytospin. Black arrows, membrane blebbing; black arrowheads, nuclear fragmentation. CDDO-Me activates the Bax protein in neuroblastoma cells. Previous studies indicated a decrease in the cytosolic level of Bax protein following exposure with CDDO-Me 12. To examine the effects of CDDO-Me on mitochondrial membrane 27 proteins and neuroblastoma viability, we first utilized the property of the 6A7 anti-Bax monoclonal antibody to bind the activated conformation of Bax protein 69, 70. SK-N-AS and 15N cells were treated for 6 hours with CDDO-Me (0 - 100 nM) (Figure 7A). Staurosporine (1 µM) was used as a positive control. Cells were harvested and activated Bax was immunoprecipitated using the 6A7 monoclonal antibody. Western blot analysis of the immunoprecipitates demonstrated an approximate 10 to 15-fold increase in activated Bax with exposure to 10 and 50 nM of CDDO-Me in SK-N-AS, respectively, and approximately 15-fold increase after treatment with 1 µM staurosporine in comparison to untreated samples. These data indicate that Bax changes conformation and becomes active at as early as 6 hours when cells are treated with as low as 10 nM of CDDO-Me (Figure 7A). Interestingly, Bax activation in 15N was only detectable at 10 nM, 6 hours after treatment with CDDO-Me and not detectable at higher concentrations of CDDO-Me or 1 µM of staurosporine (Figure 7A). This observation might reflect a complete translocation of the active form of Bax to the membrane (mitochondrial) fraction (Figure 7B) in 15N cells, which was excluded from the immunoprecipitation reaction. To monitor Bax translocation to mitochondria, we treated 15N cells with 10, 100 and 250 nM CDDO-Me or 1 µM staurosporine (Figure 7B). Adherent (alive or early apoptotic) and floating (late apoptotic, dead or mitotic) cells were collected separately 6 hours after treatment. Mitochondrial and cytosolic fractions were prepared and resolved on SDS-PAGE. Bax protein levels in all fractions were determined using Western blotting. A ~2.7 fold decrease in Bax levels was detected in the cytosolic fraction of adherent cells at doses of CDDO-Me as low as 10 nM, and a significant increase (~23 28 fold) in Bax protein was noted in the mitochondrial fraction of the floating cells at 10 nM or higher of CDDO-Me. Interestingly, there was an increase in Bax protein level in the cytosolic fraction of the floating cells (Figure 7B, top panel, lines 6, 7, 8 and 9) after treatment with CDDO-Me, but not with staurosporine. This may be due to de novo synthesis of the Bax protein, and though characteristic of 15N cells, was not observed in SK-N-AS cells. It is noteworthy that 15N cells exhibit a relatively modest response to staurosporine in comparison to CDDO-Me (Figure 7B, lanes 5 and 10). 15N cells may not activate Bax significantly in response to staurosporine (as observed in the Bax activation experiment, Figure 8A), even though the total steady-state level of Bax is increased by 6 hours after treatment with 1 µM of staurosporine (Figure 7C). To identify biomarkers of CDDO-Me activity in the treated cancer cells, we performed Western blot analyses of the whole cell extracts of 15N cells (Figure 7C), with antibodies to commonly induced stress-response proteins: HO-1, Hsp70 and Bax 71. CDDO-Me was added at 0, 60, 100 and 250 nM concentrations, cells were harvested 6 hours later and whole cell extracts were prepared. We observed as much as a 100 fold increase in the steady-state levels of HO-1 and Hsp70 at 100 and 250 nM of CDDO-Me and ~ 3 – 6 fold induction of Bax protein level at 60, 100 and 250 nM of CDDO-Me. 29 Figure 7: Exposure to CDDO-Me results in Bax confirmation change and activation in SK-NAS and 15N neuroblastoma cells. Cells were treated and harvested as described in the Methods. A: Whole cell extract was analyzed in Input and western blot analysis was performed. B: CDDO-Me induces translocation of Bax protein into mitochondria in 15N neuroblastoma cell lines. Cells were treated with various doses of CDDO-Me or staurosporine for a control for six hours. C: CDDO-Me induces steady-state levels of stress response proteins. 15N cells were treated with various doses of CDDO-Me for six hours. CDDO-Me induces dose-dependent activation of caspases and apoptosis in neuroblastoma cells. To determine whether G2/M arrest and Bax activation were coupled to caspase activation, caspases-3 and caspase-8 activities were measured in SKN-AS cells and 15N cells (data not shown), and both time course and dose response curves were determined (Figure 8A and B). Both caspase-3 and caspase-8 were activated as early as 30 minutes after treatment with 250 nM CDDO-Me. Concentrations of 10 – 30 100 nM CDDO-Me were sufficient to induce both caspase-3 and caspase-8 at the 18 hour time point. However, concentrations in the range from 250 nM – 1 µM CDDO-Me produced a greater effect (~ 6 – 20 fold induction in caspase activity). A B 25.0 Caspase3 Caspase8 30 22.5 nmols AMC released/mg protein/hr. nmols AMC released/mg protein/hr. 35 Caspase 3 Caspase 8 20.0 17.5 15.0 12.5 10.0 7.5 5.0 25 20 15 10 5 2.5 0 0.0 0 10 100 250 500 1000 Dose of CDDO-Me (nM) 0 0.5 1 3 6 18 Time (hrs.) after CDDO-Me treatment 24 Figure 8: Activation of caspases-3 and -8 in SK-N-AS neuroblastoma cells following treatment with CDDO-Me. A. Dose-dependence after six hour exposure to 0-250 nM of CDDO-Me. B. Time course 0-24 hours of caspases-3 and -8 activation by 250 nM CDDO-Me. Experiments were performed in triplicate. Error bars represent standard errors of means for each point. AMC: 7-amino-4-methoxy coumarin. CDDO-Me inhibits neuroblastoma xenograft growth in vivo. To determine the effect of orally-administered CDDO-Me on tumor growth in vivo, NIH-III mice were inoculated subcutaneously with SK-N-AS neuroblastoma cells (5x106 cells/mouse) and were then fed either control diet or diet containing CDDO-Me (100 mg/kg of diet), starting on the day of injection. Tumor volume was measured at least once each week and in both groups mice were euthanized when the tumor volume exceeded 2500 mm3, with 20 – 40 days required for tumors in the control group to reach this volume (Figure 9B). However, in mice receiving the diet with CDDO-Me, the tumors did not reach the above volume until 30 – 75 days or 1.5 – 2 times longer than in the control group (Figure 31 9C). These data demonstrate the ability of CDDO-Me to inhibit neuroblastoma growth in vivo when administered as a single agent. A. CDDO-Me (n=8) 1.2 Control (n=8) 5000 B. P=0.0099 Tumor volume (mm 3) Probability 1 0.8 0.6 0.4 4000 3000 2000 1000 0.2 0 0 0 20 40 60 0 80 20 40 60 80 Tim e post tum or injection (Days) Survival time (Days) 5000 Tumor volume (mm3) C. 4000 3000 2000 1000 0 0 20 40 60 80 Time post tumor injection (Days) Figure 9: Oral administration of CDDO-Me suppresses the growth of neuroblastoma xenografts in vivo. Animals were inoculated subcutaneously with SK-N-AS neuroblastoma cells. A. Survival fraction of untreated control versus CDDO-Me treated animals over time. B. Increase in tumor volume over time in untreated animals. C. Kinetics of tumor growth in animals treated with CDDO-Me over time. Discussion The synthetic triterpenoids are a promising class of new agents currently under development for the treatment and prevention of cancer, with CDDO-Me now in Phase I clinical trials in adults. Extensive efforts directed towards elucidating the molecular targets mediating effects on tumor cell growth and viability have uncovered the capacity 32 of these agents to modulate an array of oncogenic, tumor suppressor and stress response pathways that are also clearly involved in the pathogenesis of pediatric solid tumors. However, little or no attention has been directed at defining the activity of the triterpenoids in any preclinical model of childhood cancer. A total of 22 pediatric solid tumor cell lines were tested for their responses to CDDO-derived triterpenoids in vitro. Three different methods were used (colony formation, thymidine incorporation and RT-CES) and IC50 values derived from these studies segregated with specific methods. One set of values reflected the concentration of synthetic triterpenoid required for the inhibition of colony formation, DNA replication and cell proliferation and was in the range of 5 to 130 nM, depending on the cell line and CDDO derivative. The second set of IC50 values reflected the drug concentration required for 50% reduction in cell population determined by RT-CES. These values ranged from 110 to 630 nM for CDDO-Me in all cell lines examined. In comparison, the IC50 for the commonly used drug doxorubicin is in the range of 1.5 to 10 µM (1 - 5 µg/ml) when determined in neuroblastoma cell lines by the WST-1 assay 72. Likewise, IC50 for cisplatin in primary neuroblastoma is in the range of 0.6 – 50 µM 73, 74. It is important to note that the inhibitory activity of CDDO-Me, CDDO-TFEA, CDDO-DE, CDDO-EA and CDDO-Im is uniformly observed at nanomolar concentrations that are readily achieved in vivo without toxicity. Therefore, the synthetic triterpenoids are extremely attractive candidates to develop as potential adjuncts to standard chemotherapy agents currently in routine use for pediatric solid tumors. We observed an increase in G2/M and sub-G2/M populations following treatment with CDDO-Me (Figure 5). G2/M arrest, coupled with inhibition of BrdU and thymidine 33 incorporation during the S-phase, indicated that CDDO-Me targeted the S and G2/M phases of the cell cycle. One could interpret these data as an indicator that CDDO-Me affected processes required for DNA rearrangement during the DNA synthesis and mitotic chromosome condensation and separation. The increase in the sub-G2/M population could also be due to DNA degradation as a result of mitotic catastrophe. The accumulation of cells in sub-G2/M has been frequently observed in association with induction of apoptosis in response to stress and other anti-cancer agents 43, 75, and may represent a common response to DNA damage and other forms of cellular stress. Previous studies indicated a decrease in the cytosolic level of Bax protein following exposure with CDDO-Me 12. Within this work, changes in Bax conformation and sub-cellular distribution were investigated. Conformational activation of the Bax protein was detected using the specific 6A7 antibody, which recognizes an epitope hidden in the inactive Bax form 76, but exposed when Bax is activated by an apoptotic stimulus. As little as 10 nM CDDO-Me was sufficient to rapidly activate Bax in neuroblastoma cells, resulting in translocation of Bax from cytosol to mitochondria (Figure 7). The activation of caspase-3 and -8 by sub-micromolar doses of CDDO-Me occurred rapidly in neuroblastoma cells, demonstrating that classical mechanisms of apoptosis are induced by triterpenoids within minutes after treatment of neuroblastoma cells (Figure 8), followed by classical morphologic changes of apoptotic cell death (Figure 6). We have not detected, however, any DNA laddering (data not shown), which suggested that a calcium-dependent DNAse is not activated by CDDO-Me and is not required for triterpenoid-induced apoptosis. 34 The suppression of tumor growth in vivo by the triterpenoids could be a consequence of either direct anti-tumor effects and of indirect effects on the tumor microenvironment 68. The triterpenoids have been shown to inhibit angiogenesis in vivo 77 , which may reflect inhibition of endothelial cell proliferation, a physiological process that happens in the early stages of angiogenesis and is critical for tumor growth. HUVEC cells, which have been used as a surrogate marker for angiogenesis, are highly sensitive to CDDO-Me, with an IC50 of 110 nM after 24 hour treatment (Table 1). While our data demonstrate the ability of CDDO-Me to suppress the growth of neuroblastoma xenografts in athymic mice (Figure 9), future studies will aim to better define optimal dosing and the potential to act synergistically with standard agents and other biologic response modifiers. In the present in vivo studies with mice, dosing of CDDO-Me was limited by the intrinsic toxicity of this agent to small rodents such as rats and mice. By now, it is well established that both non-human primates and human patients are much less susceptible than rodents to any toxic effects of CDDO-Me; in 28-day studies, monkeys will tolerate oral doses of CDDO-Me that are 10 times greater than those which cause significant toxicity in rodents 78. Future Directions and Conclusions In this work, we have shown the rapid activation of caspase-8 in neuroblastoma cells that were treated with CDDO-Me. The loss of caspase-8 has also been shown to lead to resistance to drug induced apoptosis and to potentate metastasis 79 where deletion and inactivation of caspase-8 is commonly found in neuroblastomas 80, 81. What is interesting from our study is that the two cell lines which seem to be somewhat resistant 35 to CDDO-Me treatment (SK-N-SH and IMR5) do not express caspase-8. There have been some reports suggesting that caspase-8 may act as a tumor suppressor gene in neuroblastoma 81, 82 and others that link MYCN amplification to caspase-8 activity 81, 83, 84 but this remains controversial. A future study would be beneficial to understand caspase8 expression in neuroblastoma cell lines, with and without MYCN amplification, and identify how caspase-8 may mediate CDDO–induced apoptosis. Upstream of MYCN such regulators as the PI3K/Akt pathway have been also shown to play a role in tumorgenesis 85. Inhibition in the PI3K pathway components lead to MYCN destabilization 86. Akt activation is now associated with poor clinical outcomes especially in high-grade, late stage tumors 7. CDDO-Me has been shown to inhibit Akt signaling proteins in prostate cancer cells 87, 88 and it may be worth exploring CDDO’s effects on Akt signaling in neuroblastoma. Another factor that leads to tumor survival and poor prognosis in neuroblastoma is angiogenesis 89. Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and has been correlated with unfavorable histology and increased aggressive behavior in neuroblastomas 90. Known targets of CDDO, such as IKKβ and therefore inhibiting the NF-κB pathway, have been linked to suppression of angiogenesis. The inhibition of NF-κB as well as STAT meditation has been shown to play an important role in angiogenesis 91, 92. CDDO-Me has been shown to be an effective agent in suppressing angiogenesis in cell culture and in vivo where picomolar doses had a striking potency to inhibit the angiogenic effects of VEGF and tumor necrosis factor-α 77. Furthermore, it has been suggested that constitutively active NF-κB is required for survival of neuroblastoma cells 93. It would be interesting to further explore the role of 36 this inhibition in CDDO-induced apoptosis as well as angiogenesis as noted above. A better understanding of the protein expression of key genes altered in neuroblastoma or with the treatment of CDDO could help clinicians identify patients who would be most likely to have a better response to treatment with CDDO. The efficacy of the triterpenoids has been demonstrated in preclinical studies that include models of prevention or treatment in various types of cancer, either alone or in combination with conventional chemotherapy in cancers the exhibit resistance to standard agents. 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