Download human neuroblastoma cells rapidly enter cell cycle

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. Although early stage neuroblastomas are readily treatable with chemotherapy,
late stage (IV) treatment is not favorable. Stage IV tumors may initially respond to
chemotherapy but usually become increasingly resistant to the drugs and induced
apoptosis resulting in relapse. It has been shown that CDDO is able to induce apoptosis
in lines that are resistant to conventional therapies and therefore it would be worth
exploring whether or not CDDO could do the same in late stage neuroblastomas. Taken
together this work supports the concept that derivatives of CDDO are a relevant class of
new agents that should be moved into clinical development and evaluation for their
activity against pediatric solid tumors.
37
References
1.
Brodeur GM. Neuroblastoma: Biological Insights Into a Clinical Enigma. Nature
Reviews 2003; 3:203-16.
2.
Gutierrez JC, Fischer AC, Sola JE, Perez EA, Koniaris LG. Markedly improving
survival of neuroblastoma: a 30-year analysis of 1,646 patients. Pediatric Surgery
International 2007; 23:637-46.
3.
Armstrong MB, Bian X, Liu Y, Subramanian C, Ratanaproeksa AB, Shao F, Yu
VC, Kwok RP, Opipari AW, Castle VP. Signaling from p53 to NF-kappa B determines
the chemotherapy responsiveness of neuroblastoma. Neoplasia (New York, NY 2006;
8:964-74.
4.
Kuroda H, Sugimoto T, Horii Y, Sawada T. Signaling pathway of ciliary
neurotrophic factor in neuroblastoma cell lines. Medical and Pediatric Oncology 2001;
36:118-21.
5.
Lau L, Hansford LM, Cheng LS, Hang M, Baruchel S, Kaplan DR, Irwin MS.
Cyclooxygenase inhibitors modulate the p53/HDM2 pathway and enhance
chemotherapy-induced apoptosis in neuroblastoma. Oncogene 2007; 26:1920-31.
6.
Slack AD, Chen Z, Ludwig AD, Hicks J, Shohet JM. MYCN-directed centrosome
amplification requires MDM2-mediated suppression of p53 activity in neuroblastoma
cells. Cancer Research 2007; 67:2448-55.
7.
Opel D, Poremba C, Simon T, Debatin KM, Fulda S. Activation of Akt predicts
poor outcome in neuroblastoma. Cancer Research 2007; 67:735-45.
8.
Liby K, Royce DB, Williams CR, Risingsong R, Yore MM, Honda T, Gribble
GW, Dmitrovsky E, Sporn TA, Sporn MB. The synthetic triterpenoids CDDO-methyl
ester and CDDO-ethyl amide prevent lung cancer induced by vinyl carbamate in A/J
mice. Cancer Research 2007; 67:2414-9.
9.
Honda T, Rounds BV, Bore L, Finlay HJ, Favaloro FG, Jr., Suh N, Wang Y,
Sporn MB, Gribble GW. Synthetic oleanane and ursane triterpenoids with modified rings
A and C: a series of highly active inhibitors of nitric oxide production in mouse
macrophages. Journal of Medicinal Chemistry 2000; 43:4233-46.
10.
Suh N, Wang Y, Honda T, Gribble GW, Dmitrovsky E, Hickey WF, Maue RA,
Place AE, Porter DM, Spinella MJ, Williams CR, Wu G, Dannenberg AJ, Flanders KC,
Letterio JJ, Mangelsdorf DJ, Nathan CF, Nguyen L, Porter WW, Ren RF, Roberts AB,
Roche NS, Subbaramaiah K, Sporn MB. A novel synthetic oleanane triterpenoid, 2cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative,
and anti-inflammatory activity. Cancer Research 1999; 59:336-41.
38
11.
Honda T, Rounds BV, Gribble GW, Suh N, Wang Y, Sporn MB. Design and
synthesis of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, a novel and highly active
inhibitor of nitric oxide production in mouse macrophages. Bioorganic & Medicinal
Chemistry Letters 1998; 8:2711-4.
12.
Konopleva M, Tsao T, Ruvolo P, Stiouf I, Estrov Z, Leysath CE, Zhao S, Harris
D, Chang S, Jackson CE, Munsell M, Suh N, Gribble G, Honda T, May WS, Sporn MB,
Andreeff M. Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and
differentiation in acute myelogenous leukemia. Blood 2002; 99:326-35.
13.
Hyer ML, Croxton R, Krajewska M, Krajewski S, Kress CL, Lu M, Suh N, Sporn
MB, Cryns VL, Zapata JM, Reed JC. Synthetic triterpenoids cooperate with tumor
necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer
cells. Cancer Research 2005; 65:4799-808.
14.
Kim Y, Suh N, Sporn M, Reed JC. An inducible pathway for degradation of FLIP
protein sensitizes tumor cells to TRAIL-induced apoptosis. The Journal of Biological
Chemistry 2002; 277:22320-9.
15.
Samudio I, Konopleva M, Hail N, Jr., Shi YX, McQueen T, Hsu T, Evans R,
Honda T, Gribble GW, Sporn M, Gilbert HF, Safe S, Andreeff M. 2-Cyano-3,12dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) directly targets mitochondrial
glutathione to induce apoptosis in pancreatic cancer. The Journal of Biological Chemistry
2005; 280:36273-82.
16.
Chauhan D, Li G, Podar K, Hideshima T, Shringarpure R, Catley L, Mitsiades C,
Munshi N, Tai YT, Suh N, Gribble GW, Honda T, Schlossman R, Richardson P, Sporn
MB, Anderson KC. The bortezomib/proteasome inhibitor PS-341 and triterpenoid
CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome
bortezomib resistance. Blood 2004; 103:3158-66.
17.
Chintharlapalli S, Papineni S, Konopleva M, Andreef M, Samudio I, Safe S. 2Cyano-3,12-dioxoolean-1,9-dien-28-oic acid and related compounds inhibit growth of
colon cancer cells through peroxisome proliferator-activated receptor gamma-dependent
and -independent pathways. Molecular Pharmacology 2005; 68:119-28.
18.
Liby K, Voong N, Williams CR, Risingsong R, Royce DB, Honda T, Gribble
GW, Sporn MB, Letterio JJ. The synthetic triterpenoid CDDO-Imidazolide suppresses
STAT phosphorylation and induces apoptosis in myeloma and lung cancer cells. Clin
Cancer Res 2006; 12:4288-93.
19.
Robert M. Kliegman ea. Nelson Textbook of Pediatrics. Elsevier Inc, 2007.
20.
Schwab M, Shimada, H, Joshi, V, Brodeur, GM. In: Pathology and Genetics of
Tumours of the Nervous System, Kleihues, P, Cavenee, WK Neuroblastic tumours of
adrenal gland and sympathetic nervous system. France, 2000.
39
21.
Brodeur GM CR. Neuroblastoma. In: Principles and Practices of Pediatric
Oncology. Philadelphia, PA: Lippencott-Raven, 1997.
22.
Institue. NIoHNC. Sympathetic Nervous System Tumors.
23.
Kushner BH, Gilbert F, Helson L. Familial neuroblastoma. Case reports, literature
review, and etiologic considerations. Cancer 1986; 57:1887-93.
24.
Perri P, Longo L, McConville C, Cusano R, Rees SA, Seri M, Conte M, Romeo
G, Devoto M, Tonini GP. Linkage analysis in families with recurrent neuroblastoma.
Annals of the New York Academy of Sciences 2002; 963:74-84.
25.
Brodeur GM, Green AA, Hayes FA, Williams KJ, Williams DL, Tsiatis AA.
Cytogenetic features of human neuroblastomas and cell lines. Cancer Research 1981;
41:4678-86.
26.
Gilbert F, Balaban G, Moorhead P, Bianchi D, Schlesinger H. Abnormalities of
chromosome 1p in human neuroblastoma tumors and cell lines. Cancer Genetics and
Cytogenetics 1982; 7:33-42.
27.
Norris MD, Bordow SB, Marshall GM, Haber PS, Cohn SL, Haber M. Expression
of the gene for multidrug-resistance-associated protein and outcome in patients with
neuroblastoma. The New England Journal of Medicine 1996; 334:231-8.
28.
Srivatsan ES, Ying KL, Seeger RC. Deletion of chromosome 11 and of 14q
sequences in neuroblastoma. Genes, Chromosomes & Cancer 1993; 7:32-7.
29.
Gilbert F, Feder M, Balaban G, Brangman D, Lurie DK, Podolsky R, Rinaldt V,
Vinikoor N, Weisband J. Human neuroblastomas and abnormalities of chromosomes 1
and 17. Cancer Research 1984; 44:5444-9.
30.
Look AT, Hayes FA, Nitschke R, McWilliams NB, Green AA. Cellular DNA
content as a predictor of response to chemotherapy in infants with unresectable
neuroblastoma. The New England journal of medicine 1984; 311:231-5.
31.
Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of
N-myc in untreated human neuroblastomas correlates with advanced disease stage.
Science New York, NY 1984; 224:1121-4.
32.
Ribatti D, Raffaghello L, Pastorino F, Nico B, Brignole C, Vacca A, Ponzoni M.
In vivo angiogenic activity of neuroblastoma correlates with MYCN oncogene
overexpression. International Journal of Cancer 2002; 102:351-4.
33.
Lutz W, Stohr M, Schurmann J, Wenzel A, Lohr A, Schwab M. Conditional
expression of N-myc in human neuroblastoma cells increases expression of alpha-
40
prothymosin and ornithine decarboxylase and accelerates progression into S-phase early
after mitogenic stimulation of quiescent cells. Oncogene 1996; 13:803-12.
34.
Kohl NE, Gee CE, Alt FW. Activated expression of the N-myc gene in human
neuroblastomas and related tumors. Science New York, NY 1984; 226:1335-7.
35.
Nisen PD, Waber PG, Rich MA, Pierce S, Garvin JR, Jr., Gilbert F, Lanzkowsky
P. N-myc oncogene RNA expression in neuroblastoma. Journal of the National Cancer
Institute 1988; 80:1633-7.
36.
Kang JH, Rychahou PG, Ishola TA, Qiao J, Evers BM, Chung DH. MYCN
silencing induces differentiation and apoptosis in human neuroblastoma cells.
Biochemical and Biophysical Research Communications 2006; 351:192-7.
37.
Nara K, Kusafuka T, Yoneda A, Oue T, Sangkhathat S, Fukuzawa M. Silencing
of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell
differentiation in a neuroblastoma cell line with MYCN amplification. International
Journal of Oncology 2007; 30:1189-96.
38.
Philip A. Pizzo DGP. Principles and Practice of Pediatric Oncology 2002.
39.
Phillips DR, Rasbery JM, Bartel B, Matsuda SP. Biosynthetic diversity in plant
triterpene cyclization. Current Opinion in Plant Biology 2006; 9:305-14.
40.
Nishino H, Nishino A, Takayasu J, Hasegawa T, Iwashima A, Hirabayashi K,
Iwata S, Shibata S. Inhibition of the tumor-promoting action of 12-Otetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds. Cancer
Research 1988; 48:5210-5.
41.
Huang MT, Ho CT, Wang ZY, Ferraro T, Lou YR, Stauber K, Ma W, Georgiadis
C, Laskin JD, Conney AH. Inhibition of skin tumorigenesis by rosemary and its
constituents carnosol and ursolic acid. Cancer Research 1994; 54:701-8.
42.
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, Sutter TR,
Baumgartner KJ, Roebuck BD, Liby KT, Yore MM, Honda T, Gribble GW, Sporn MB,
Kensler TW. Potent protection against aflatoxin-induced tumorigenesis through induction
of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)dien-28-oyl]imidazole. Cancer Research 2006; 66:2488-94.
43.
Temmink OH, Hoebe EK, Fukushima M, Peters GJ. Irinotecan-induced
cytotoxicity to colon cancer cells in vitro is stimulated by pre-incubation with
trifluorothymidine. Eur J Cancer 2007; 43:175-83.
44.
Ito Y, Pandey P, Sporn MB, Datta R, Kharbanda S, Kufe D. The novel
triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by
a caspase-8 dependent mechanism. Molecular Pharmacology 2001; 59:1094-9.
41
45.
Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420:860-7.
46.
Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in
the initiation and promotion of malignant disease. Cancer Cell 2005; 7:211-7.
47.
Dinkova-Kostova AT, Liby KT, Stephenson KK, Holtzclaw WD, Gao X, Suh N,
Williams C, Risingsong R, Honda T, Gribble GW, Sporn MB, Talalay P. Extremely
potent triterpenoid inducers of the phase 2 response: correlations of protection against
oxidant and inflammatory stress. Proceedings of the National Academy of Sciences of the
United States of America 2005; 102:4584-9.
48.
Couch RD, Browning RG, Honda T, Gribble GW, Wright DL, Sporn MB,
Anderson AC. Studies on the reactivity of CDDO, a promising new chemopreventive and
chemotherapeutic agent: implications for a molecular mechanism of action. Bioorganic &
Medicinal Chemistry Letters 2005; 15:2215-9.
49.
Talalay P, Dinkova-Kostova AT, Holtzclaw WD. Importance of phase 2 gene
regulation in protection against electrophile and reactive oxygen toxicity and
carcinogenesis. Advances in Enzyme Regulation 2003; 43:121-34.
50.
Yu X, Kensler T. Nrf2 as a target for cancer chemoprevention. Mutation Research
2005; 591:93-102.
51.
Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense
mechanisms against electrophiles and reactive oxygen species. Advances in Enzyme
Regulation 2006; 46:113-40.
52.
Nguyen T, Yang CS, Pickett CB. The pathways and molecular mechanisms
regulating Nrf2 activation in response to chemical stress. Free Radical Biology &
Medicine 2004; 37:433-41.
53.
Ji Y, Lee HJ, Goodman C, Uskokovic M, Liby K, Sporn M, Suh N. The synthetic
triterpenoid CDDO-imidazolide induces monocytic differentiation by activating the Smad
and ERK signaling pathways in HL60 leukemia cells. Molecular Cancer Therapeutics
2006; 5:1452-8.
54.
Alabran JL, Cheuk A, Liby K, Sporn M, Khan J, Letterio J, Leskov KS. Human
neuroblastoma cells rapidly enter cell cycle arrest and apoptosis following exposure to C28 derivatives of the synthetic triterpenoid CDDO. Cancer Biology & Therapy 2008;
7:709-17.
55.
Liby KT, Yore MM, Sporn MB. Triterpenoids and rexinoids as multifunctional
agents for the prevention and treatment of cancer. Nature Reviews 2007; 7:357-69.
56.
Lapillonne H, Konopleva M, Tsao T, Gold D, McQueen T, Sutherland RL,
Madden T, Andreeff M. Activation of peroxisome proliferator-activated receptor gamma
42
by a novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces
growth arrest and apoptosis in breast cancer cells. Cancer Research 2003; 63:5926-39.
57.
Dragnev KH, Pitha-Rowe I, Ma Y, Petty WJ, Sekula D, Murphy B, Rendi M, Suh
N, Desai NB, Sporn MB, Freemantle SJ, Dmitrovsky E. Specific chemopreventive agents
trigger proteasomal degradation of G1 cyclins: implications for combination therapy.
Clin Cancer Res 2004; 10:2570-7.
58.
Zou W, Liu X, Yue P, Zhou Z, Sporn MB, Lotan R, Khuri FR, Sun SY. c-Jun
NH2-terminal kinase-mediated up-regulation of death receptor 5 contributes to induction
of apoptosis by the novel synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1, 9dien-28-oate in human lung cancer cells. Cancer Research 2004; 64:7570-8.
59.
Zou W, Chen S, Liu X, Yue P, Sporn MB, Khuri FR, Sun SY. c-FLIP
Downregulation Contributes to Apoptosis Induction by the Novel Synthetic Triterpenoid
methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate (CDDO-Me) in Human Lung
Cancer Cells. Cancer Biology & Therapy 2007; 6.
60.
Pedersen IM, Kitada S, Schimmer A, Kim Y, Zapata JM, Charboneau L, Rassenti
L, Andreeff M, Bennett F, Sporn MB, Liotta LD, Kipps TJ, Reed JC. The triterpenoid
CDDO induces apoptosis in refractory CLL B cells. Blood 2002; 100:2965-72.
61.
Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, Chiao PJ,
Achanta G, Arlinghaus RB, Liu J, Huang P. Selective killing of oncogenically
transformed cells through a ROS-mediated mechanism by beta-phenylethyl
isothiocyanate. Cancer Cell 2006; 10:241-52.
62.
Shishodia S, Sethi G, Konopleva M, Andreeff M, Aggarwal BB. A synthetic
triterpenoid, CDDO-Me, inhibits IkappaBalpha kinase and enhances apoptosis induced
by TNF and chemotherapeutic agents through down-regulation of expression of nuclear
factor kappaB-regulated gene products in human leukemic cells. Clin Cancer Res 2006;
12:1828-38.
63.
Yue P, Zhou Z, Khuri FR, Sun SY. Depletion of intracellular glutathione
contributes to JNK-mediated death receptor 5 upregulation and apoptosis induction by
the novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate
(CDDO-Me). Cancer Biology & Therapy 2006; 5:492-7.
64.
Ahmad R, Raina D, Meyer C, Kharbanda S, Kufe D. Triterpenoid CDDO-Me
blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179. The Journal
of Biological Chemistry 2006; 281:35764-9.
65.
Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer
development and progression. Nature Reviews 2005; 5:749-59.
43
66.
Yore MM, Liby KT, Honda T, Gribble GW, Sporn MB. The synthetic
triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole blocks nuclear
factor-kappaB activation through direct inhibition of IkappaB kinase beta. Molecular
Cancer Therapeutics 2006; 5:3232-9.
67.
Letterio JJ. TGF-beta signaling in T cells: roles in lymphoid and epithelial
neoplasia. Oncogene 2005; 24:5701-12.
68.
Albini A, Sporn MB. The tumour microenvironment as a target for
chemoprevention. Nat Rev Cancer 2007; 7:139-47.
69.
Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and
Bcl-X(L) during apoptosis. Proceedings of the National Academy of Sciences of the
United States of America 1997; 94:3668-72.
70.
Chitambar CR, Wereley JP, Matsuyama S. Gallium-induced cell death in
lymphoma: role of transferrin receptor cycling, involvement of Bax and the
mitochondria, and effects of proteasome inhibition. Molecular Cancer Therapeutics 2006;
5:2834-43.
71.
Crosby LM, Hyder KS, DeAngelo AB, Kepler TB, Gaskill B, Benavides GR,
Yoon L, Morgan KT. Morphologic analysis correlates with gene expression changes in
cultured F344 rat mesothelial cells. Toxicology and Applied Pharmacology 2000;
169:205-21.
72.
Hamasaki K, Kogure K, Ohwada K. A biological method for the quantitative
measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a watersoluble tetrazolium salt. Toxicon 1996; 34:490-5.
73.
Woessmann W, Chen X, Borkhardt A. Ras-mediated activation of ERK by
cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell
lines. Cancer Chemother Pharmacol 2002; 50:397-404.
74.
Rosenhagen MC, Soti C, Schmidt U, Wochnik GM, Hartl FU, Holsboer F, Young
JC, Rein T. The heat shock protein 90-targeting drug cisplatin selectively inhibits steroid
receptor activation. Mol Endocrinol 2003; 17:1991-2001.
75.
Sadikovic B, Rodenhiser DI. Benzopyrene exposure disrupts DNA methylation
and growth dynamics in breast cancer cells. Toxicol Appl Pharmacol 2006; 216:458-68.
76.
Peyerl FW, Dai S, Murphy GA, Crawford F, White J, Marrack P, Kappler JW.
Elucidation of some Bax conformational changes through crystallization of an antibodypeptide complex. Cell Death Differ 2007; 14:447-52.
44
77.
Vannini N, Lorusso G, Cammarota R, Barberis M, Noonan DM, Sporn MB,
Albini A. The synthetic oleanane triterpenoid, CDDO-methyl ester, is a potent
antiangiogenic agent. Molecular Cancer Therapeutics 2007; 6:3139-46.
78.
Kral R MC AR. Rodent and Primate Toxicity Studies of Oral RTA 402 (CDDOMe), A Novel Agent with Anti-cancer and Anti-mucositis Activities. 2005.
79.
Stupack DG, Teitz T, Potter MD, Mikolon D, Houghton PJ, Kidd VJ, Lahti JM,
Cheresh DA. Potentiation of neuroblastoma metastasis by loss of caspase-8. Nature 2006;
439:95-9.
80.
Takita J, Yang HW, Bessho F, Hanada R, Yamamoto K, Kidd V, Teitz T, Wei T,
Hayashi Y. Absent or reduced expression of the caspase 8 gene occurs frequently in
neuroblastoma, but not commonly in Ewing sarcoma or rhabdomyosarcoma. Medical and
Pediatric Oncology 2000; 35:541-3.
81.
Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG,
Look AT, Lahti JM, Kidd VJ. Caspase 8 is deleted or silenced preferentially in childhood
neuroblastomas with amplification of MYCN. Nature Medicine 2000; 6:529-35.
82.
Fulda S, Kufer MU, Meyer E, van Valen F, Dockhorn-Dworniczak B, Debatin
KM. Sensitization for death receptor- or drug-induced apoptosis by re-expression of
caspase-8 through demethylation or gene transfer. Oncogene 2001; 20:5865-77.
83.
Gonzalez-Gomez P, Bello MJ, Lomas J, Arjona D, Alonso ME, Aminoso C,
Lopez-Marin I, Anselmo NP, Sarasa JL, Gutierrez M, Casartelli C, Rey JA. Aberrant
methylation of multiple genes in neuroblastic tumours. relationship with MYCN
amplification and allelic status at 1p. Eur J Cancer 2003; 39:1478-85.
84.
Cui H, Li T, Ding HF. Linking of N-Myc to death receptor machinery in
neuroblastoma cells. The Journal of Biological Chemistry 2005; 280:9474-81.
85.
Johnsen JI, Segerstrom L, Orrego A, Elfman L, Henriksson M, Kagedal B,
Eksborg S, Sveinbjornsson B, Kogner P. Inhibitors of mammalian target of rapamycin
downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in
vivo. Oncogene 2008; 27:2910-22.
86.
Chesler L, Schlieve C, Goldenberg DD, Kenney A, Kim G, McMillan A, Matthay
KK, Rowitch D, Weiss WA. Inhibition of phosphatidylinositol 3-kinase destabilizes
Mycn protein and blocks malignant progression in neuroblastoma. Cancer Research
2006; 66:8139-46.
87.
Deeb D, Gao X, Dulchavsky SA, Gautam SC. CDDO-me induces apoptosis and
inhibits Akt, mTOR and NF-kappaB signaling proteins in prostate cancer cells.
Anticancer Research 2007; 27:3035-44.
45
88.
Deeb D, Gao X, Dulchavsky SA, Gautam SC. CDDO-Me inhibits proliferation,
induces apoptosis, down-regulates Akt, mTOR, NF-kappaB and NF-kappaB-regulated
antiapoptotic and proangiogenic proteins in TRAMP prostate cancer cells. Journal of
Experimental Therapeutics & Oncology 2008; 7:31-9.
89.
Meitar D, Crawford SE, Rademaker AW, Cohn SL. Tumor angiogenesis
correlates with metastatic disease, N-myc amplification, and poor outcome in human
neuroblastoma. J Clin Oncol 1996; 14:405-14.
90.
Langer I, Vertongen P, Perret J, Fontaine J, Atassi G, Robberecht P. Expression
of vascular endothelial growth factor (VEGF) and VEGF receptors in human
neuroblastomas. Medical and Pediatric Oncology 2000; 34:386-93.
91.
Albini A, Noonan DM, Ferrari N. Molecular pathways for cancer
angioprevention. Clin Cancer Res 2007; 13:4320-5.
92.
Chen Z, Han ZC. STAT3: A Critical Transcription Activator in Angiogenesis.
Medicinal Research Reviews 2008; 28:185-200.
93.
Bian X, Opipari AW, Jr., Ratanaproeksa AB, Boitano AE, Lucas PC, Castle VP.
Constitutively active NFkappa B is required for the survival of S-type neuroblastoma.
The Journal of Biological Chemistry 2002; 277:42144-50.
46