The Role of ASK1 in Arsenic Trioxide-induced
Cell Death
Stanley Kwan
Experimental Medicine
McGill University, Montreal
November, 2012
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Masters of Science.
© Stanley Kwan 2012
Table of Contents
Abstract
Résumé
Acknowledgements
Contribution of Authors
Abbreviations
Chapter 1: Literature Review
1. Introduction to Cancer
1. Treatment modalities
2. Acute Promyelocytic Leukemia
1. Genetic Basis and Pathogenesis
2. Treatment Options
3. Arsenic Trioxide as a Therapeutic Agent
1. Physical and Chemical properties of Arsenic and Arsenic
Compounds
2. Toxicity of Arsenic Compounds
1.Environmental Contamination
2.Arsenic Metabolism
3.Health Risks to Arsenic Exposure
3. Chronology of Arsenic as a Therapeutic Agent
4. ATO in the Treatment of Acute Promyelocytic Leukemia
1.Clinical Responses to ATO in Acute Promyelocytic
Leukemia Patients
2.Adverse Effects of ATO treatment
5. Use of ATO in Other Malignancies
1.Multiple Myeloma
2.Myelodysplastic Syndrome
3.Chronic Myelogenous Leukemia
4.Solid tumors
4. Mechanisms of ATO Signaling in APL
1. Induction of Partial Differentiation
2. Induction of Apoptosis
1.Intrinsic Pathway
2.Extrinsic Pathway
3. Reactive Oxygen Species Generation
1.Mechanism of ATO-induced Reactive Oxygen
Species Accumulation
2.Reactive Oxygen Species Signaling
3.Role of Antioxidants in Resistance to ATO-induced
Cell Death
4.Role of Pro-oxidants in Potentiating ATO-induced
Cell Death
4. NFκB Inhibition
5. Activation of Mitogen-Activated Protein Kinases
1.Role of JNK Activation in ATO-induced Apoptosis
2.SEK1 Activation
1
3
5
7
8
9
11
11
11
12
13
13
14
14
15
15
16
16
17
19
20
20
21
22
22
23
23
24
24
25
26
26
27
28
28
29
30
30
31
31
32
3.JNK Targets
4.Signal Transduction from ATO-induced
Generation to JNK Activation
6. AKT Pro-survival Targets
5. Role of ASK1 in ATO-Induced Apoptosis
1. Mechanism of ASK1 Activation
1.Role in Stress-Induced Cell Death
2.Activation of ASK1
2. Other roles of ASK1
6. Combination Therapies
Chapter 2: Role of ASK1 in ATO-induced Apoptosis
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
Chapter 3: Regulation of ASK1 in ATO-induced Apoptosis
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
Chapter 4: Conclusion
References
2
33
ROS
33
33
34
35
35
35
35
38
39
39
42
48
60
65
65
68
71
77
80
81
ABSTRACT
Arsenic trioxide (ATO) is an effective treatment for acute promyelocytic leukemia
(APL). While it is undergoing clinical trials for numerous malignancies including
multiple myeloma, myelodysplastic syndrome, lymphoma and solid tumors, it has
demonstrated only limited efficacy as a single agent. However, it may hold
promise as part of a combination therapy.
Thus investigation to elucidate the
mechanisms of action underlying these clinical responses may lead to generation
of rational combination therapies to increase its therapeutic spectrum. Previous
work has described a pathway required for ATO-induced apoptosis in APL cells
involving the generation of reactive oxygen species (ROS), and the subsequent
induction of a specific mitogen-activated protein kinase (MAPK) cascade that
includes both stress-activated protein kinase (SAPK)/ERK kinase 1 (SEK1) and cJun N-terminal kinases (JNK) activation.
However, the link between ROS
production and activation of SEK1 remains to be elucidated. Apoptosis signaling
kinase 1 (ASK1) is a MAP3K upstream of SEK1 that has been implicated in the
induction of stress-induced signaling.
Using murine embryonic fibroblasts (MEFs) derived from ASK1-/- mice; we
provide evidence that ASK1 is a strong mediator for ATO-induced apoptosis and
JNK activation. In an APL cell line, we show that ATO activates ASK1 in a
dose- and time-dependent manner. However, knockdown of ASK1 in APL cells
enhanced susceptibility to ATO, undergoing apoptosis and growth inhibition more
than their wild type counterparts.
The same impact was observed in the
knockdown of SEK1, a direct downstream MAP2K of ASK1, and the knockdown
of ASK1 in MCF-7 cells, a human breast cancer cell line. This led us to postulate
that ASK1 had a pro-apoptotic function in non-transformed fibroblasts, but was
pro-survival in malignant cells. Indeed, transformation of ASK1-/- MEFs restored
their sensitivity to ATO-induced apoptosis and growth inhibition. Taken together,
these results suggest that ASK1 can have both pro-apoptotic and anti-apoptotic
roles depending on the transformation state of the cells.
3
One model of ASK1 regulation suggests that ASK1 is kept in an inactive form by
reduced thioredoxin-1 (Trx1). During oxidative stress, Trx1 is oxidized and
releases ASK1 for activation.
Immunoprecipitation of ASK1 followed by
immuoblotting for Trx1 in APL cells shows a strong basal association that is lost
with ATO treatment.
Furthermore, the activity of thioredoxin reductase 1
(TrxR1), an enzyme that converts oxidized Trx1 into reduced Trx1, is
significantly decreased following ATO treatment.
This suggests that ATO
activates ASK1 signaling by ROS-mediated oxidation of Trx1 and by maintaining
Trx1 in its oxidized state by decreasing TrxR1 activity. In addition, we show that
inhibition of TrxR1 with the TrxR1 inhibitor Auranofin sensitizes APL cells to
ATO-induced apoptosis. Overall, our results suggest that targeting Trx1 may
enhance ATO-induced apoptosis in a novel combination therapy.
4
RÉSUMÉ
L’arsenic trioxyde (ATO) est un traitement efficace contre la leucémie
promyélocitaire aïgue (APL). Alors qu’il est testé dans le cadre de plusieurs
essais cliniques sur différents cancers comme le myélome multiple, le syndrome
myélodysplasique, le lymphome et les tumeurs solides, il ne montre qu’une
efficacité limitée en tant qu’agent unique. Quoiqu’il en soit, il pourrait être
prometteur au sein d’une thérapie combinée.
Ainsi, l’étude des mécanismes
d’action responsables des bénéfices cliniques apportés par l’arsenic trioxide
pourrait mener à la mise au point de nouvelles thérapies combinées afin d’élargir
le spectre thérapeutique d’ATO. Des travaux antérieurs ont décri une voix de
signalisation, requise pour l’induction de l’apoptose par l’arsenic dans des cellules
d’APL, qui implique la génération d’espèces actives de l’oxygène (ROS), ainsi
que l’induction subséquente d’une cascade de protéine kinases mitogène-activées
(MAPK) spécifique qui inclut à la fois la protéine kinase stress-activée
(SAPK)/ERK kinase 1(SEK1) ainsi que l’activation de la kinase c-jun N-terminal
(JNK). Néanmoins, le lien entre la production de ROS et l’activation de SEK1
reste à être élucidé. La kinase de signalisation de l’apoptose (ASK1) est une
MAP3K en amont de SEK1 qui a été impliquée dans l’induction de la
signalisation induite par le stress.
En utilisant des fibroblastes d’embryions de souris (MEFs) dérivées de souris
ASK-/-, nous montrons qu’ASK1 est un médiateur fort de l’apoptose et de
l’activation de JNK par ATO. Dans une lignée cellulaire APL, nous montrons
qu’ATO active ASK1 en fonction du temps et de la dose. Par contre, une sousrégulation d’ASK1 dans des cellules APL augmente la susceptibilité envers ATO,
ce qui se traduit par une diminution de la croissance et une augmentation de
l’apoptose par rapport aux cellules APL sauvages. Un impact similaire est observé
lors d’une sous régulation de SEK1, une MAP2K directement en aval d’ASK1,
ainsi que lors d’une sous régulation d’ASK1 dans des cellules MCF-7, des
cellules de cancer du sein humain. Cela nous a conduit à postuler qu’ASK1 a une
fonction pro-apoptotique dans les fibroblastes non-transformés, mais anti5
apoptotique dans des cellules malignes. En effet, la transformation des cellules
MEFs ASK-/- a restauré leur sensibilité envers l’arrêt de la croissance et
l’apoptose induits par ATO, ce qui souligne les différences entre les cellules
normales et les cellules transformées. Dans l’ensemble, ces résultats suggèrent
qu’ASK1 a un rôle important dans la sensibilité à l’ATO qui dépend du contexte.
Un des schémas de régulation d’ASK1 suggère qu’ASK1 est séquestré sous une
forme inactive par la thioredoxine-1 réduite (Trx1). Durant le stress oxydatif,
Trx1 est oxydée et libère ASK1 afin qu’il puisse être activé. L’immunoprécipitation d’ASK1 suivie d’un immuno-marquage for Trx1 dans des cellules
APL montre une association basale forte qui est perdue lors du traitement avec
ATO. De plus, l’activité de la réductase de la thioredoxine (TrxR1), une enzyme
qui convertit Trx1 oxydée en Trx1 réduite, est diminuée de façon significative
après traitement avec ATO. Cela suggère qu’ATO active la signalisation d’ASK1
en oxydant Trx1, via les ROS, ainsi qu’en inhibant la réduction de Trx1 en
diminuant l’activité de TrxR1. De plus, nous montrons que l’inhibiteur de TrxR1,
Auranofin, sensibilise les cellules APL à l’apoptose induite par ATO. Cela
suggère que la régulation d’ASK1 dépend du statut redox de Trx1. Dans
l’ensemble, nos résultats suggèrent que le fait de cibler Trx1 pourrait augmenter la
signalisation d’ASK1 et l’apoptose induite par ATO au sein d’une nouvelle
thérapie combinée.
6
ACKNOWLEDGEMENTS
I would like to take this opportunity to highlight all the wonderful people who
have supported me throughout my two years. My sincerest acknowledgement
goes to my supervisor Dr. Koren Mann, who has provided me with the
opportunity to work in her laboratory as a graduate student. Her enthusiasm,
expertise, constructive criticism, and guidance were always welcomed and have
enabled me to become a better scientist. I would like to thank Nicolas Garnier for
teaching me the tools and skills of the laboratory, as well as his continued
guidance. He also did a wonderful job on the translation of the abstract. My
sincere gratitude goes to Sonia del Rincon for helping me trouble shoot through
the most difficult surprises science had to offer. I want to thank Alexander Kelly
for being there to share the graduate student experience with me through all the
highs and lows. I am also grateful to Dr. Wilson H. Miller Jr. and all the past and
present members of the Mann/Miller lab for their continued support, friendship
and assistance: Felicia Villasenor, Jessica Nichol, Monica Dobocan, Cynthia
Guilbert, Manuel Flores Molina, Myrian Colombo, Andy Petruccelli, Bonnie
Huor, Moqing Wang, , Torsten Nielsen, Maryse Lemaire, Alicia Bolt, Filippa
Pettersson, Lu Yao, Audrey Emond, Daphné Dupéré-Richer, Mena Kinal,
Flamingo Tang, Sarah Chau, Yao Zhan, Genevieve Redstone, Ingrid Tam, Lydie
Barbeau,
Jules
Eustache,
and
Thomas
Di
Lenardo.
I also want to acknowledge the tremendous support I have received from my
friends and family without whom this manuscript would not be possible.
7
CONTRIBUTIONS OF AUTHORS
The thesis is composed of 4 chapters prepared in accordance to the guidelines
outlined by the department of Graduate and Postdoctoral Studies.
The
contribution of each author described below.
Chapter 2
The majority of the experiments were completed by Stanley Kwan.
Myrian
Colombo completed Figures 1B, 1C and 2.
Chapter 3
The majority of the experiments were completed by Stanley Kwan. Nicolas
Garnier completed Figure 4.
8
ABBREVIATIONS
ASK1 = Apoptosis signal-regulating kinase 1
AML = Acute myelogenous leukemia
AMS = 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid
Ang II = Angiotensin II
APL = Acute promyelocytic leukemia
AQP = Aquaporin
ATO = Arsenic trioxide
ASCT = Autologous stem cell transplantation
ATRA = All-trans retinoic acid
ATF-2 = Activating transcription factor-2
ATM = Sodium aurothiomalate
BSO = Buthionine sulfoximine
CML = Chronic myeloid leukemia
CCC = C-terminal coiled-coil
CREB = cAMP response element-binding protein
DAR = S-dimethylarsino-glutathione
DTNB = 5,5'-di-thio-bis-(2-nitrobenzoic acid)
ERK1/2 = Extracellular signal-regulated kinase 1/2
FAB = French-American-British
FKHR = Forkhead transcription factor
GPx = Glutathione peroxidase
GSH = Glutathione (γ-glutamylcysteinylglycine)
HDCT = High-dose chemotherapy
IKK = IKβ kinase
JNK = c-jun N-terminal kinase
MAPK = Mitogen-activated protein kinase
MAPKK = MAPK kinase
MAPKKK = MAPK kinase kinase
MEF = Mouse embryonic fibroblast
MDS = Myelodysplastic syndrome
9
MM = Multiple myeloma
MRP1/ABCC1 = Multidrug resistance protein 1
NAC = N-acetyl-cysteine
NF-κB = Nuclear factor kappa B
NPM = Nucleophosmin
NuMA = Nuclear matrix
PLZF = Promyelocytic leukemia zinc finger
PML = Promyelocytic leukemia gene
PARP = poly (ADP-ribose) polymerase
PP5 = Protein phosphatase 5
PTPC = Permeability transition pore complex
RARɑ = Retinoic acid receptor acid receptor ɑ
ROS = Reactive oxygen species
SAPK = Stress-activated protein kinase
SEK1 = Stress-enhanced kinase 1
SOD = Super-oxide dismutase
SRB = Sulforhodamine-B
TNF = Tumor necrosis factor
TRAFs = Tumor necrosis factor-ɑ receptor-associated factors
Trx1 = Thioredoxin 1
TrxR1 = Thioredoxin reductase 1
10
CHAPTER 1: LITERATURE REVIEW
1. Introduction to Cancer
Cancer defines a broad range of diseases comprised of abnormal cells that divide
in a dysregulated fashion and can spread to other tissues through the blood
circulation and lymphatic system. It can be categorized broadly into carcinoma,
sarcoma, leukemia, lymphoma, myeloma, and central nervous system diseases.1
The six classical hallmarks of cancer are resisting cell death, sustaining
proliferative signaling, evading growth suppressors, activating invasion and
metastasis, enabling replicative immortality and inducing angiogenesis.2
Cancer is a worldwide problem and is a leading cause of death. In Canada, over
500 Canadians are diagnosed with cancer every day while 200 die from it.3 The
number of individuals diagnosed with cancer continues to rise, with prostate
cancer being the most commonly diagnosed cancer in men and breast cancer
being the most commonly diagnosed cancer in women. However, the leading
cause of cancer death in both men and women is lung cancer.
While only a small proportion of cancers are attributed to genetic defects (5-10%),
the majority are greatly influenced by environmental and lifestyle factors. 4 These
include and are not limited to age, cigarette smoking, alcohol, sun exposure,
environmental pollutants, infections, stress, obesity, physical inactivity, hormones
and diet.4,5 Age is the most important risk factor for developing cancer and will
remain a significant risk factor due to the aging population.5
1.1. Treatment modalities
Conventional treatment modalities include surgery, radiation, chemotherapy,
hormone therapy, and biological therapy.5,6 These modalities are often used in
combination depending on the type and location of the cancer, the extent of
cancer spread, the patient’s age and general health, among other factors. In the
11
case of leukemia, choice of treatment depends mainly on the type of leukemia,
age and the presence of leukemic cells in the cerebrospinal fluid and is limited by
the unavailability of surgery.7 Patients with acute leukemia require immediate
treatment with the goal of first achieving remission and then preventing relapse
with consolidation therapy leading to a cure.7 On the other hand, patients with
chronic leukemia are rarely cured and may require stem cell transplants.
2. Acute Promyelocytic Leukemia
Acute promyelocytic leukemia (APL) is a clinically distinct subtype of acute
myelogenous
leukemia
promyelocytes due to
(AML)
characterized
by
the
accumulation
a block in granulocytic differentiation.8
of
It is
morphologically classified by the French-American-British (FAB) classification
as AML-M3. Patients with the disease experience a high tendency for severe
bleeding due to fibrinogenopenia and disseminated intravascular coagulation.9
Some features of APL are illustrated in figure 1.1.
Figure 1.1: (Adapted from Liu et al)10 Features of acute promyelocytic
leukemia. (A) Severe bleeding and (B) accumulation of abnormal promyelocytes
in the bone marrow of an APL patient.
12
2.1. Genetic Basis and Pathogenesis
Cytogenetically, APL is characterized most often by a reciprocal translocation
between chromosomes 15 and 17 resulting in the fusion of the promyelocytic
leukemia (PML) gene and the retinoic acid receptor ɑ (RARɑ) gene.11 RARɑ is a
ligand-dependent transcription factor stimulated by all-trans retinoic acid
(ATRA).12 RARɑ signaling is normally required for the transcription of genes
necessary for myeloid differentiation. However, in APL, the resulting aberrant
PML/RARɑ oncoprotein binds to DNA with higher affinity and recruits corepressors and histone deacetylases to silence and block the transcription of target
genes necessary in myeloid differentiation.12,13 While the majority of APL cases
are attributed to the translocation t(15:17), a small subset of APL can be caused
by other gene arrangement combinations involving the fusion of the RARɑ gene
to other partners, including promyelocytic leukemia zinc finger (PLZF),14
nucleophosmin (NPM)15 and nuclear matrix (NuMA).12,16 In all scenarios, RARɑ
signaling prevents normal myeloid development.
2.2. Treatment Options
Until the early 1980s, treatment of APL consisted of cytotoxic chemotherapy
treatment, usually with anthracycline in combination with cytosine arabinoside,
similar to the treatment of other AML subtypes.9,17 However, a study conducted
by a Chinese group demonstrated that all-trans retinoic acid (ATRA), a derivative
of vitamin A, was effective in APL patients; especially those refractory to
conventional treatment.17,18 This finding changed the traditional paradigm for
cancer treatment from strictly cytotoxic chemotherapy to induction of cellular
differentiation as a feasible means of treatment. Since then, the efficacy of ATRA
has been validated through numerous studies and it is now used as first line
therapy to treat APL patients.19 Despite the successes of ATRA, relapse and
acquired resistance is common in patients, necessitating other therapeutics. The
void is filled by arsenic trioxide (ATO), a compound that has shown remarkable
13
efficacy in treatment of patients with APL, especially those who have relapsed
and are refractory to ATRA treatment.20 The chemical structures of the ATRA
and ATO are illustrated in Figure 1.2.
Figure 1.2 (Adapted from Liu et al)10 Chemical structure of ATRA (left) and
arsenic trioxide (right)
3. Arsenic Trioxide as a Therapeutic Agent
3.1. Physical and Chemical properties of Arsenic and Arsenic Compounds
Arsenic is a group V metalloid possessing chemical properties intermediate
between metals and non-metals.21 It is found naturally in more than 200 different
mineral forms and is present in the earth’s crust with an abundance of 5 mg/kg.22
Each form has its unique physical, chemical, and toxicological properties.23
Anthropogenic sources further increase exposure to arsenic, raising concerns of
pollution in soil and groundwater.22
Arsenic can assume several oxidation states but there are two in particular that are
biologically important and are associated with its cytotoxic potential: +3 and +5
with the +3 state being more potent.21 In addition, it also exists as inorganic and
organic forms.21 The three major inorganic forms are arsenic trisulfide (As2S3),
arsenic disulfide (As2S2) and arsenic trioxide (As203) also known as yellow, red
and white arsenic respectively (Figure 1.3).24
14
A) Arsenic Trisulfide
B) Arsenic Disulfide
C) Arsenic Trioxide
(As2S3)
(As2S2)
(As203)
Red Arsenic
White Arsenic
Yellow Arsenic
Orpiment
25
26
Realgar
Arsenolite27
Figure 1.3 Types of inorganic arsenic
3.2. Toxicity of Arsenic Compounds
3.2.1. Environmental Contamination
Arsenic is found naturally in the atmosphere, soils, rocks, and water and is
circulated into the environment through natural means and anthropogenic
activities.28 It poses a health risk predominantly when present in food and
drinking water, where chronic exposure can lead to serious health problems in
many parts of the world.28,29 In India and Bangladesh alone, there has been an
estimated 60-100 million people who are at risk of disease from drinking arseniccontaminated water.22 The accumulating reports of the toxicological effects of
arsenic in drinking water prompted the World Health Organization to limit the
acceptable concentration of arsenic in drinking water to 10 ug per liter as of
1993.28 However, many developing countries still lack the resources to adequately
meet these safety guidelines.
15
3.2.2.
Arsenic Metabolism
Arsenic can be absorbed into the body through various means but ingestion and
gastrointestinal absorption of inorganic arsenic is the most common path.
Ingested arsenic is rapidly absorbed into the blood from the gastrointestinal tract
and then subsequently transported into the body cells through aquaporins
(AQP).23,30 This occurs predominantly in the liver. However, different forms of
arsenic distribute differently in the body and have varying toxicities.31 The liver is
the
primary
site
of
arsenic
metabolism
where
arsenic
undergoes
biotransformation, a series of biochemical reactions that convert a compound into
a more water-soluble compound to facilitate excretion.32 This process includes
many steps including reductions and oxidative methylations to form pentavalent
organic metabolites.33 The intermediates in the process, particularly the
methylated intermediates, are more toxic than inorganic arsenic.34 Arsenic is then
excreted predominantly in the urine and feces.
3.2.3. Health Risks to Arsenic Exposure
Arsenic has been recognized for centuries as having significant acute toxicity and
chronic toxicity following exposure. Acute exposure can cause death and has
been used as an intentional poison throughout history as a murder weapon.
Chronic toxicity is caused by long term exposure to low doses of arsenic and has
stimulated more concern due to the increasing levels of environmental arsenic.35
Millions of people suffer from the effects of chronic arsenic poisoning which is
now regarded as an international public health problem that needs to be
addressed.35
Arsenic toxicity depends on a variety of factors. Soluble inorganic arsenicals are
generally more toxic than organic ones;35-37 however, there is evidence that
methylated organic ATO metabolites are more potent growth inhibitors and
apoptotic inducers than inorganic arsenicals.38 Our lab has already shown that S16
dimethylarsino-glutathione (DAR), an
organic
arsenical
synthesized by
conjugating dimethylarsenic to glutathione, is more potent than ATO in inducing
oxidative stress and apoptosis.39 Furthermore, the trivalent form of arsenic is more
toxic than the pentavalent form due to its ability to strongly bind thiol groups (SH) of proteins with a high cysteine content.37,40
Acute exposure to large doses of arsenic can yield symptoms as early as 30
minutes starting with burning lips and dysphagia.35 This may be followed by
violent vomiting and eventually hematemesis. Gastrointestinal symptoms are also
readily present and may lead to electrolyte imbalance, hypertension and hypoxia.
Multi-organ failure can ensue, causing death.35,36,41
Chronic exposure to arsenic has been implicated in numerous cancerous
conditions, including strong evidence for the development of lung,42 bladder43 and
skin cancer.35,36 Epidemiologic studies are underway to provide evidence for the
development of other cancers as well. Chronic arsenic exposure has also been
linked to skin disease, diabetes mellitus, hypertension, cardiovascular disease,
perturbed porphyrin metabolism and irreversible non-cirrhotic portal hypertension
among others.35,36,41 The symptoms of chronic exposure differ among individuals,
population groups and geographical areas.44
3.3. Chronology of Arsenic as a Therapeutic Agent
While widely recognized for its role as a poison, arsenic paradoxically has an
equally enriching history as a medicine and has been used for more than 2400
years in this capacity.45 The major milestones of arsenic usage are illustrated in
Figure 1.4. Hippocrates was thought to have used arsenic-containing minerals
realgar and orpiment pastes to treat ulcers. Dioscorides later recognized that ATO
could serve as a therapeutic when given at low doses in the treatment of
tuberculosis and emphysema. Concurrently, arsenic was used in traditional Asian
folk medicines to treat ailments such as malaria and fever.
17
Arsenic gained popularity in Europe in the 17th century when William Withering
advocated for arsenic-based therapies by justifying that medicines are simply
poisons but used at low doses.45 Over a century later, Thomas Fowler created a
therapeutic of potassium bicarbonate-based solution of arsenic that was used to
treat a wide variety of ailments. Fowler’s solution gained the nickname “the
mule” for its strength and efficacy.46 It was reported to have an anti-leukemic
effect but was eventually replaced by radiation therapy.47 By 1910, Paul Ehrlich
was using an organic-based arsenic therapeutic called salversan to treat
tuberculosis and syphilis.48
With the advent of other medicines including antibiotics, the use of arsenic
declined. It made a resurgence through the help of a published report showing
good responses in patients with chronic myeloid leukemia (CML) to ATO.49
However, these patients later developed chronic arsenic poisoning, exhibiting
symptoms including skin pigmentation, skin keratosis, liver cirrhosis, polyneuritis
and gastrointestinal problems.50
The major breakthrough came for arsenic in the 1970s by studies conducted at the
Harbin Medical University in China where they carefully monitored cancer
patients treated with intravenous infusions of Ailing 1, a crude ATO solution.51,52
The treatment showed tremendous efficacy in APL patients and was carefully
followed up by clinical trials in Shanghai Second Medical University, Europe and
the United States.53 These studies confirmed ATO as an effective therapeutic in
APL relapsed patients, with a disease that is refractory to ATRA treatment.
18
Figure 1.4 (Adapted from Zhu et al., 2002)45 Timeline of arsenic as a therapeutic
agent
3.4. ATO in the Treatment of Acute Promyelocytic Leukemia
The successes of ATO in the clinical trials in China, Europe and the United States
led to the approval of the drug for treatment of relapsed APL by the U.S. Food
and Drug Administration (FDA) in September 2000 under the brand name,
TrisenoxTM.47 However, it was not certain whether ATO would be equally
effective in patients with newly diagnosed APL. A Chinese group from the
Shanghai Institute of Hematology studied and compared the responses to ATO in
relapsed and newly diagnosed APL patients and observed similar rates of
remission in both groups.54 However, they showed that ATO induced significant
19
hepatic toxicity in the newly diagnosed APL patients and subsequently
recommended ATO for use only in maintenance or consolidation therapy
following remission by ATRA. Since then, ATO in newly diagnosed patients has
been further studied in combination with standard ATRA/chemotherapy to
reinforce the efficacy of the regimen.55,56 Ravandi et al. and Shen et al have
shown that the combination of ATRA and ATO in newly diagnosed APL patients
is an effective and safe substitute for conventional chemotherapy–containing
regimens and either ATRA or ATO alone.57,58 Furthermore, ATO has been
studied as a substitute for induction and consolidation regimen to minimize
cytotoxicity.
3.4.1. Clinical Responses to ATO in Acute Promyelocytic Leukemia
Patients
Lengfelder et al. recently consolidated the results from all the clinical studies of
ATO in treating relapsed APL and newly diagnosed APL.56 Overall, 15 studies
and 304 patients were included in the evaluation of ATO in patients with relapsed
APL after first line ATRA and chemotherapy treatment. The responses to ATO
treatment were significantly positive, with 86% of the patients achieving complete
remission while only 7% showed an inadequate response and another 7% died
from induction therapy. In comparison, a total of 314 of 362 (87%) of the patients
from the studies using ATO in newly diagnosed APL patients achieved a
complete response with comparable toxicities.
3.4.2. Adverse Effects of ATO treatment
While many anti-cancer drugs and chemotherapies elicit significant side effects,
ATO has relatively few significant side effects. Nearly half the APL relapsed
patients treated with ATO experienced some form of mild side effects including
fatigue, fever, edema, nausea, anorexia, diarrhea, emesis, headache, insomnia,
20
cough,
dyspnea,
dermatitis,
tachycardia,
pain,
hypomagnesemia
and
hyperglycemia.20,53,59
Leukocytosis has been reported in 50% of the patients, likely due to the
differentiation of all the APL blasts at once as a direct effect on the leukemic
cells, but could spontaneously resolve without chemotherapy.20,53,54 However, in
25-50% of the cases, it can develop into retinoic acid syndrome with
accompanying fever, skin rash, and edema.60 These symptoms are usually
alleviated by administration of corticosteroids.
The most common serious symptoms occurring in more than 10% of the patients
include
abdominal
pain,
epistaxis,
dyspnea,
hypoxia,
bone
pain,
thrombocytopenia, neutropenia, hypokalemia and hyperglycemia.20,53,59 Other rare
side effects include distal muscular dystrophy, cardiac dysrhythmias, prolonged
QT intervals, and ventricular premature contractions.59,61-63
3.5. Use of ATO in Other Malignancies
The relatively rare appearance of significant side effects compounded with its
high efficacy in the treatment of relapsed APL patients stimulated interest to
extend ATO into other cancer settings, particularly in hematological
malignancies. Preclinical studies confirmed that ATO showed some efficacy
against multiple malignant cell lines.64 Currently, there have been 96 completed or
ongoing clinical trials with ATO as a single agent and in combinations, in a
variety of malignancies including metastatic melanoma, acute myeloid leukemia,
multiple myeloma, central nervous system tumors, myelodysplastic syndrome,
hepatocellular carcinoma, chronic lymphocytic leukemia, colorectal cancer, breast
cancer, and lymphoma among others.65 Lessons learned from these studies have
shown that while ATO has limited efficacy as a single agent in settings outside of
APL, it may hold promise when used as part of a combination therapy.
21
3.5.1. Multiple Myeloma
Multiple myeloma (MM) is an incurable hematological malignancy characterized
by an accumulation of monoclonal plasma cells in the bone marrow.66,67 There are
more than 15 000 newly diagnosed cases in the United States alone and has a
median survival of 3-4 years.68
Currently, patients with MM are treated with high-dose chemotherapy (HDCT),
thalidomide, bortezomib, lenaldomide and autologous stem cell transplantation
(ASCT) but patients eventually relapse and become refractory to conventional
treatment.67,68
When used as a monotherapy, numerous studies have shown that ATO can elicit a
response in up to 33% of MM patients, where a response is defined as a greater
than 25% reduction in serum M protein.69,70 The results in combination therapies
with Bortezomib and ascorbic acid have been more encouraging; showing
enhanced clinical activity against relapsed and refractory MM and is well
tolerated.71,72
3.5.2. Myelodysplastic Syndrome
Myelodysplastic syndrome (MDS) is a heterogeneous hematological malignancy
characterized by a hyper-proliferative bone marrow, dysplasia of the cellular
elements and ineffective hematopoiesis.73 Much of the morbidity and mortality is
attributed to severe anemia, bleeding and infections.
The only curative method is high-dose chemotherapy with allogeneic
transplantation.
The disease still remains a challenge, particularly in elderly
74
patients. Preclinical studies have shown that ATO can induce apoptosis in MDS
cell lines at clinically achievable concentrations.74 Results from two phase II
multicenter trials demonstrated that ATO treatment yielded an overall rate of
22
hematological improvement of 29% with one complete and one partial
response.75,76 ATO is still undergoing evaluation for routine use in MDS patients.
3.5.3. Chronic Myelogenous Leukemia
Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm resulting
from a balanced reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem
cells leading to the fusion of the c-abl oncogene and the break point cluster
region.77 The resulting Bcr-Abl oncoprotein is a constitutively activated tyrosine
kinase that promotes the unregulated growth and accumulation of myeloid cells in
the bone marrow and in the blood circulation.78
Many patients treated with conventional therapy with the tyrosine kinase inhibitor
Imatinib have poor outcomes. This is highlighted by the 60% of patients who fail
to achieve a complete response after 18 months on therapy.79,80 Resistance can
develop due to the inability of Imatinib to completely inhibit Bcr-Abl and the
overexpression of Bcr-Abl.80 ATO may play a role in down-regulating Bcr-Abl
and is being investigated in combination with Imatinib to treat patients with
Imatinib resistance.81 Thus far, no significant responses have been observed
although the regimen is well tolerated.66
3.5.4. Solid tumors
ATO has shown promising preclinical activity in solid tumor cancer cell lines by
inhibiting proliferation, decreasing cell viability and inducing apoptosis.82 These
cancer cell lines include prostate cancer, bladder cancer, ovarian cancer, colon
cancer, gastric cancer, cervical cancer and esophageal cancer cell lines. This led
to numerous clinical trials that are underway including in advanced metastatic
melanoma,83,84 hepatocellular carcinoma,85 and advanced head and neck cancer,86
but has thus far only shown limited efficacy compared to in APL.
23
4. Mechanisms of ATO Signaling in APL
In recent years, the clinical successes of ATO in APL have stimulated
investigation in understanding the mechanism of action of ATO. Initially, it was
shown that arsenic-resistant APL cells underwent apoptosis in addition to being
susceptible to
ATO-induced partial differentiation, suggesting different
mechanisms of ATO action.87 Currently, the working model suggests that ATO
has dose dependent dual effects on APL cells, inducing apoptosis at higher
concentrations (0.5 – 2 µM) and partial differentiation at lower concentrations
(0.1 – 0.5 µM).88
4.1. Induction of Partial Differentiation
Similarly to ATRA treatment, ATO is capable of inducing the degradation of the
PML-RARɑ fusion protein to release the maturation block and promote
differentiation in APL cells.89,90 In normal cells, the PML protein is found in
macromolecular structures in the nucleus called nuclear bodies.91,92 In APL cells,
the PML nuclear bodies exhibit a nuclear microspeckle pattern with a
corresponding loss of PML function. PML is a tumor suppressor that antagonizes
many processes for the initiation and development of malignancy, including
growth arrest and apoptosis.91 In APL cells, the PML-RARɑ fusion oncoprotein
blocks the expression of genes necessary for normal differentiation. Upon ATO
treatment at low concentrations (0.1 – 0.5 μM), PML and PML-RARɑ are
targeted to nuclear bodies, sumoylated, polyubiquitinated by the SUMOdependent ubiquitin ligase RNF4, and then degraded by the proteasome.89,93
The PML-RARɑ in APL cells recruits and constitutively binds co-repressors
SMART/N-CoR and histone deacetylases that promotes the differentiation block
through inhibiting the expression of genes necessary for normal myeloid
differentiation.94 ATO can release the differentiation block by interfering with the
24
ability of the PML-RARɑ fusion protein to recruit the co-repressors thereby
promoting the expression of genes necessary for myeloid differentiation.9,95
However, the presence of the PML-RARɑ fusion protein does not contribute
entirely to the exquisite sensitivity of APL cells to ATO. Work in our lab has
shown that the sensitivity of APL cells to ATO is not due to the expression and
regulation of PML-RARɑ.96,97
4.2. Induction of Apoptosis
ATO in high concentrations (0.5 – 2 µM) has been shown to induce apoptosis in
APL and non-APL hematological malignancy and solid tumor cell lines.88
Through numerous studies, many mechanisms are implicated in ATO-induced
apoptosis including the generation of reactive oxygen species (ROS),98 decrease
in the mitochondrial membrane potential ΔΨm,
99
caspase cascade activation,100
down-regulation of Bcl-2 expression,101 cell cycle arrest,102 and the opening of the
permeability transition pore complex (PTPC) in the mitochondrial membrane
through direct interaction.103
Apoptosis is an evolutionarily well-conserved regulated programmed cell death
that occurs in different physiological and pathological situations.104 It is a
necessary process to control cell number and tissue size as well as maintaining
homeostasis by removing aberrant cells.
It is characterized by specific
morphological and biochemical features distinct from necrosis including cell
shrinkage, nuclear DNA fragmentation and membrane blebbing.
The central executioners of apoptosis are a family of highly well-conserved
intracellular cysteine proteases called caspases.104 They possess an active site that
allows them to cleave substrates at specific sites.
Initially expressed as a
zymogen, they require cleavage at specific sites to become activated. Caspase
activation occurs in a systematic manner where one caspase cleaves and activates
25
the next in the cascade. This allows for an efficient method to amplify and
integrate pro-apoptotic signals.
4.2.1. Intrinsic Pathway
The mitochondria is regarded as the forum of death where pro-apoptotic proteins
including cytochrome c are sequestered.104 Pro-apoptotic signals can converge on
the mitochondria and induce the depolarization of the mitochondria membrane
potential to open up the PTPC.
The PTPC is a multi-protein complex that
interacts with the apoptosis regulating protein Bcl-2/Bax family.105,106 ATO has
been reported to decrease the mitochondrial membrane potential ΔΨm and to
directly bind to the thiol groups of the PTPC and cause the release of cytochrome
c and other pro-apoptotic proteins into the cytoplasm.64,103 ATO further
contributes to apoptosis by down-regulating the expression of Bcl-2, a known
antagonist to the opening of the PTPC.101,103
In the cytoplasm, cytochrome c binds and activates Apaf-1 which then activates
procaspase-9 to begin the caspase cascade leading to cell death.107,108 At the
bottom of the cascade, caspase 3 is activated and subsequently cleaves key
substrates in the cell to result in the cellular and biochemical features of apoptosis.
Among the cleavage events is the inactivation of poly (ADP-ribose) polymerase
(PARP) which is thought to prevent the depletion of NAD and ATP, necessary
components for the later events of apoptosis.109,110
4.2.2. Extrinsic Pathway
The extrinsic death pathway employs the role of death receptors which are
members of the tumor necrosis factor (TNF) receptor gene superfamily that
transmit the death signal from the cell surface to intracellular signaling pathways
involving first the activation of caspase 8.111 Kitamura et al. reported that ATO
can contribute to inducing apoptosis through the activation of the extrinsic
26
pathway of apoptosis.112 In their study, they demonstrated that ATO activated
caspase 8 and Bid in APL cells but not in ATO-resistant APL cells. Furthermore,
inhibition of caspase 8 blocked the activation of caspase 3 and the depolarization
of the mitochondrial membrane potential. They also showed that their ATOresistant cells had up-regulated expression of glutathione (GSH) and
pharmacologic inhibition of GSH in these cells restored sensitivity to ATO,
caspase 8 activation, and depolarization of mitochondrial membrane potential.
Overall, their results suggested that ATO activates extrinsic death pathway
signaling in a ROS-dependent manner.
4.3. Reactive Oxygen Species Generation
The accumulation of ROS plays a significant role in ATO-induced
cytotoxicity.36,98 ROS is important in many biochemical and physiological
processes and its up-regulation has been associated with many cancers.113 It may
also be responsible for the sensitivity to certain drugs and contribute to the
regulation of cellular proliferation, DNA damage and genomic instability.
ROS are oxygen-containing chemical species called radicals that have the ability
to exist independently.114 The extremely high chemical reactivity of these species
is attributed to the possession of unpaired electrons in their outermost orbitals.115
Oxygen possesses two electrons on its outermost orbital, rendering it extremely
susceptible to the formation of radicals. Examples of ROS includes the oxygen
molecule, superoxide radicals, O2* and the hydroxyl radical, OH*.
In eukaryotic cells under physiological conditions, most of the ROS is generated
from the mitochondrial electron transport chain where approximately 2% of the
oxygen is converted into ROS.116,117 Other ROS generating sites in the cell
includes the microsomal cytochrome P450, plasma membrane NAD/NADPH
systems and peroxisomes.114 Their formation is catalyzed by oxidation/reduction
27
reactions by enzymes such as dehydrogenases, oxidases, dioxygenases and
hydroxylases.
ROS can cause damage to all classes of macromolecules including DNA, RNA,
proteins and lipid components leading to inhibition of cellular proliferation,
apoptosis, or necrosis.118 The damage can occur through altering the redox
potential of proteins, binding proteins to other macromolecules and altering the
activity of transcription factors and many enzymes among other functions. Some
of the enzymes altered include protein kinases and phosphatases.114 Due to the
adverse effects of excess ROS accumulation, it has been under scrutiny for the
cause of cellular aging, carcinogenesis, malignant transformation and many
chronic diseases.
4.3.1. Mechanism
of
ATO-induced
Reactive
Oxygen
Species
Accumulation
The accumulation of ROS as a requirement for ATO-induced apoptosis is well
documented from studies in our lab and others.36,96,98 While the exact mechanism
remains to be elucidated, many targets have been implicated in this process.
These targets include the activation of NADPH oxidase and NO synthase,
perturbation of the mitochondrial electron transport chain and the inhibition of
antioxidant
peroxidase.
enzymes
including
thioredoxin
reductase
and
glutathione
119-121
4.3.2. Reactive Oxygen Species Signaling
ATO-induced ROS accumulation may regulate many different intracellular
signaling pathways.120 Among these pathways include the stress-induced mitogenactivated protein kinase (MAPK) signaling cascades.
Activation of the
extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade mediates
signals for growth and differentiation by activating downstream transcription
28
factors such as c-Myc and Elk1.120 ATO has also been shown to activate the
SAPK/JNK which can enter into the nucleus and regulate the activation of
activating transcription factor-2 (ATF-2) and c-Jun transcription factor to mediate
apoptosis.122 Furthermore, the p38 MAPK has been reported to be activated by
ATO and is involved in the regulation of transcription factors such as ATF-2 and
cAMP response element-binding protein (CREB), leading to conflicting outcomes
including cell proliferation and apoptosis.120 One report combining ATO and a
p38-MAPK inhibitor show enhanced ATO-induced apoptosis and growth
inhibition in APL and multiple myeloma cells.123,124
4.3.3. Role of Antioxidants in Resistance to ATO-induced Cell Death
Given the damaging effects of excess ROS in cells, cells must rely on intracellular
defense mechanisms to mitigate the threat. One mechanism of protection makes
use of anti-oxidant enzymes including super-oxide dismutase (SOD), and
catalase.125 While SOD metabolises superoxides, catalase metabolizes hydrogen
peroxide to reduce ROS stress.126
Cells also possess reductive-oxidative buffering systems to regulate the levels of
ROS. The glutathione system involves the enzyme glutathione peroxidase (GPx)
which uses reduced glutathione to catalyze the reduction of hydrogen peroxide.127
Published data from our lab has shown that pharmacologic depletion of
glutathione in ATO-resistant APL cells re-sensitizes them to ATO.96 The unique
sensitivity of APL cells to ATO can also be attributed to the lower level of basal
cellular glutathione content and therefore less buffering against ROS.127
Furthermore, the decreased level of cellular glutathione may lead to arsenic
accumulation because the arsenic cannot be glutathionylated and exported via the
multidrug resistance protein 1 (MRP1/ABCC1) transporter.128 The thioredoxin
(Trx) ROS buffering system involves the capacity of reduced Trx to become
oxidized to help maintain proteins in their reduced state.129,130 Reduced Trx can be
29
restored by the enzyme thioredoxin reductase (TrxR). We have preliminary data
demonstrating that inhibition of TrxR enhances ATO-induced cell death.
4.3.4. Role of Pro-oxidants in Potentiating ATO-induced Cell Death
Since ROS accumulation is an important mediator of ATO-induced apoptosis,
manipulation of the redox environment in cells is an attractive strategy to enhance
ATO sensitivity, particularly in non-APL settings with a high ROS buffering
capacity. APL cells are uniquely sensitive to ATO in part due to their relatively
lower GSH levels compared to other malignant cell lines.127 Indeed our lab has
already shown that the pharmacologic depletion of GSH by buthionine
sulfoximine (BSO) can synergize with ATO to induce apoptosis in a variety of
malignant cell lines including ATO-resistant APL cells and diffuse large B-cell
lymphoma.98 When ATO is combined with a pro-oxidant such as ascorbic acid,
cytotoxicity is increased in chronic lymphocytic leukemia,131,132 multiple
myeloma,133 and CML cell lines.134 The use of bryostatin 1, an analog of phorbol
myristate acetate, has been shown to synergize with ATO to enhance ROS
generation and cell killing of leukemic cells through the stimulation of NADPH
oxidase activity.119 Our lab has also demonstrated that the widely known
antioxidant trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a
vitamin E analog, can synergize with ATO to increase intracellular oxidative
stress and enhance cell death in APL, myeloma, and breast cancer cells.135
4.4. NFκB Inhibition
ATO can also promote cell death by inhibition of nuclear factor kappa B (NF-κB)
activation. NF-κB is a transcription factor that mediates the expression of prosurvival genes in APL cells and has an important role in many malignant
diseases.136 Some of the target genes include those encoding cell adhesion
molecules, pro-inflammatory cytokines, stress response enzymes, growth factors
and anti-apoptotic enzymes.137 NF-κB is normally sequestered by Iκβɑ but can be
30
activated by the IKβ kinase (IKK) complex-mediated phosphorylation and
degradation of Iκβɑ in response to extracellular signals.138 ATO has been reported
to bind to the Cys-179 residue in the activation loop of the IKKα/β subunit of
IKK, thereby inhibiting it.139 This results in the inhibition of the NF-κB cell
survival signals and leads to enhanced cell death.
4.5. Activation of Mitogen-Activated Protein Kinases
ATO-induced ROS has been shown to regulate the evolutionarily well-conserved
mitogen-activated protein kinase (MAPK) signaling cascade which is involved in
many intracellular processes including gene expression, cell proliferation, cell
survival, apoptosis, differentiation and apoptosis.140 MAPK signaling cascades are
composed of different tiers which are activated sequentially through
phosphorylation. Typically, a MAPK kinase kinase (MAP3K) activates a MAPK
kinase (MAP2K) which then in turn activates a MAPK to propagate the
intracellular signal. Each tier is subject to its own regulation.
4.5.1. Role of JNK Activation in ATO-induced Apoptosis
While several different MAPK signaling cascades exist, the activation of the
SAPK/JNK pathway is most strongly implicated in ATO-induced apoptosis. This
pathway is normally activated in response to environmental stresses including UV
light, endotoxin, inflammation and tumor necrosis factor (TNF) to mediate proapoptotic signals from the mitochondria.141 JNK is a MAPK that is activated upon
phosphorylation by upstream kinases MKK4 (SEK1) and MKK7 (SEK2).142,143
Upon activation, JNK phosphorylates and regulates transcription factors including
activating transcription factor-2 (ATF-2) and c-Jun.141
Work done in our lab has demonstrated that APL cells exhibit significant ATOinduced cell death that is preceded by ROS generation and JNK activation.97
However, ATO-resistant cell lines with up-regulated GSH content showed only
31
limited JNK activation at the same ATO concentrations. JNK activation and
ATO-sensitivity could be restored in the ATO-resistant APL cell lines by
pharmacologic depletion of GSH. Furthermore, pharmacologic inhibition of JNK
reduced sensitivity to ATO in APL cells. These results suggest that ATO-induced
ROS generation and JNK activation are required to induce apoptosis. Other
groups have also shown that JNK activation is important in ATO-induced
apoptosis in other leukemic and lymphoma cell lines including U937, Namalwa,
Jurkat, and NKM-1.144-146
There has been conflicting reports on the role of p38 activation in ATO-induced
apoptosis. In one instance, p38 was shown to have a negative regulatory role in
ATO-induced apoptosis. Pharmacologic inhibition of p38 or its direct upstream
activators Mkk3 and Mkk6 resulted in enhanced ATO-induced JNK activation
and cell death in various leukemic cell lines.123,147 However, p38 activation has
also been implicated in ATO-induced apoptosis. Kang and Lee reported that
ATO induced ROS generation and apoptosis through the activation of p38 in
HeLa cells.148 Furthermore, Iwarma et al. demonstrated that p38 activation played
a pro-apoptotic role while JNK activation played an anti-apoptotic role in U937
cells treated with unconventionally high concentrations of ATO.149 The
conflicting results may be attributed to the different cell types used and the
concentrations of ATO administered.
4.5.2. SEK1 Activation
Further evidence suggest that ATO-induced apoptosis is dependent on JNK
activation and signaling.
Published data from our lab has shown that JNK
activation is abrogated in stress-enhanced kinase 1 (SEK1) deficient mouse
embryonic fibroblasts (MEF) compared to their wild-type counterparts with a
corresponding reduced sensitivity to ATO-induced cell death.97 SEK1 is a
MAP2K that directly phosphorylates and activates JNK in response to stress
signals.
32
4.5.3. JNK Targets
JNK signaling has been reported to be able to induce the expression of genes for
both cell survival and cell death, but only sustained JNK activation mediates proapoptotic signaling.
Such is the case for treatment with ATO where JNK
activation is sustained for at least 24 hours.97 Upon activation, JNK
phosphorylates and activates a wide variety of proteins that mediate pro-apoptotic
gene expression including c-jun, Elk-1 and ATF2.150 JNK activation may also
induce apoptosis via a transcription-independent mechanism by activation of the
intrinsic death pathway through the Bax subfamily of Bcl-2-related proteins151
and through the phosphorylation of 14-3-3 proteins to regulate nuclear targeting
of c-Abl in apoptotic responses.152
4.5.4.
Signal Transduction from ATO-induced ROS Generation to JNK
Activation
ATO is transported inside the cell through aquaporins.30,153 Once inside the cell,
the exact mechanism of signal transduction from oxidative stress generation to
JNK activation is not entirely clear. JNK activation has been reported to require
small GTP-binding proteins Ras,154 Rac,155 Cdc42155 and Rho156 however; some
evidence suggests only Rac, Cdc42 and Rho are implicated in ATO-induced JNK
activation.157
4.5.5. AKT Pro-survival Targets
ATO-induced JNK activation and apoptosis is highly regulated in many
components of the signaling pathway. One of the key regulators is AKT, a
serine/threonine protein kinase that mediates cell survival signals through the
phosphorylation and inactivation of pro-apoptotic proteins including BAD158,
caspase-9159 and forkhead (FKHR) family of transcription factors.160 AKT has
33
been reported to suppress the p38 and JNK pathways through the inactivation of
the SEK1161 and the upstream apoptosis signal-regulating kinase 1 (ASK1).162
Furthermore, it may also bind to the JNK pathway scaffold proteins JIP1 163 and
POSH164 to prevent the assembly of the JNK signaling complex. Our lab has
shown that in response to ATO, AKT protein levels and activity are decreased,
correlating with JNK activation and sensitivity to ATO.165
Since AKT negatively regulates ATO-induced apoptotic pathways at many
different points, inhibition of AKT may enhance ATO-induced apoptosis. In one
report, pharmacologic inhibitors of AKT potentiated ATO-induced apoptosis in
myeloid leukemic cells through glutathione depletion and enhanced ROS
accumulation.166
5. Role of ASK1 in ATO-Induced Apoptosis
Our lab has begun defining the pathway of ATO-induced apoptosis involving the
MAPK cascade. We have shown that ROS accumulation is required and that JNK
activation is necessary for maximum induction of ATO-induced apoptosis.
Furthermore, genetic deficiency of the MAP2K, SEK1, resulted in abrogated
ATO-induced JNK activation and apoptosis. Upstream of SEK1, there are several
MAPKKK that are implicated in the induction of the SEK/JNK pathway.
However, since stress-induced signaling is strongly implicated in ASK1
activation, we hypothesized that ASK1 mediates ATO-induced stress signaling
and apoptosis.
ASK1 is a ubiquitously expressed MAPKKK that activates both JNK and p38
signaling pathways and is a strong mediator of apoptotic signals.167 It
phosphorylates and activates downstream MAPKKs including SEK1 and SEK2 in
response to cellular stress such as oxidative stress, endoplasmic reticulum stress,
calcium overload and receptor mediated inflammatory signals.168-170 While
recognized for its pro-apoptotic role, ASK1 may have other functions including
34
mediating differentiation and survival signals depending on the cell type and
context.171-173
5.1. Mechanism of ASK1 Activation
5.1.1. Role in Stress-Induced Cell Death
In response to various cytotoxic stresses, the MAPK cascade is activated to relay
cell death signals terminating in p38 and JNK activation. However, there are
more than 11 different mammalian MAPKKKs that have been implicated in the
activation of JNK and/or p38 signaling.150 ASK1 has been shown to be important
in this process where ASK1 deficient (ASK1-/-) murine embryonic fibroblasts
(MEFs) are resistant to ROS and TNF-induced apoptosis.174 TNF is a proinflammatory cytokine that regulates inflammation, proliferation and apoptosis in
a ROS-dependent mechanism.
5.1.2.
Activation of ASK1
ASK1 exerts is activity as part of a high molecular complex referred to as the
ASK1 signalosome.
The formation of the signalosome requires the homo-
oligomerization of ASK1 through its C-terminal coiled-coil (CCC) domain
conjugated to other proteins.175,176 In response to stress signals, the homooligomerized ASK1 becomes activated through the phosphorylation of threonine
838 within the activation segment in the kinase domain. Activated ASK1 can
then phosphorylate and activate downstream MAPKK in the MAPK pathway.
5.2. Other roles of ASK1
While ASK1 is recognized for its pro-apoptotic role, several lines of evidence
suggest that it may also have other functions in response to stress. ASK1 has
been reported to mediate signals for differentiation and survival in the rat
35
pheochromocytoma cell line PC12172 and keratinocytes.177 It has also been
implicated in the regulation of erythroid differentiation.178
ASK1 may also contribute to diseases like cardiac hypertrophy through stressinduced signaling.179 There is evidence to suggest that ROS generation induced by
angiotensin II (Ang II) plays an important role in the intracellular signaling
response in cardiac hypertrophy.180 ASK1 may mediate G-protein-coupled
receptor agonist-induced NFκB activation to induce cardiac hypertrophy.173
Taken together, we can begin to paint a working model of ATO signaling in
inducing apoptosis (Figure 1.5).
36
Figure 1.5 Proposed ATO-induced JNK signaling pathway
37
6. Combination therapies
While ATO has been demonstrated to be an effective therapy in APL, dose
limiting toxicities limit its success in other settings when used as a single agent.
However, it may hold promise when used in combination therapies therefore it is
important to develop sensitization strategies to increase the cytotoxic effects of
the drug. One strategy requires understanding the mechanisms of ATO-induced
apoptosis to identify potential targets for combination therapies. This will allow
the generation of rational combination therapies to increase its therapeutic
spectrum and is the objective of this project.
38
CHAPTER 2: ROLE OF ASK1 IN ATO-INDUCED APOPTOSIS
Introduction
Arsenic trioxide (ATO) is an arsenic derivative that has important anti-tumor
properties. It has been shown to be an effective treatment for acute promyelocytic
leukemia (APL) especially in patients that have relapsed and have become
refractory to all-trans retinoic acid (ATRA) treatment.181,182 ATO-induced antileukemic activity is a result of dose-dependent dual effects, comprising of partial
differentiation through the degradation of PML/RARɑ at low concentrations (0.1
– 0.5 µM) and inducing apoptosis at high concentrations (0.5 – 2 µM).88
The relatively rare appearance of significant side effects compounded with its
high efficacy in the treatment of relapsed APL patients stimulated interest to
extend ATO into other cancer settings, particularly in hematological
malignancies. Currently, there have been 96 completed or ongoing clinical trials
with ATO as a single agent and in combinations in a variety of malignancies
including metastatic melanoma, acute myeloid leukemia, multiple myeloma,
central nervous system tumors, myelodysplastic syndrome, hepatocellular
carcinoma, chronic lymphocytic leukemia, colorectal cancer, breast cancer, and
lymphoma among others.65 However, ATO demonstrated limited efficacy in nonAPL settings due to dose-limiting toxicities. While ATO has limited efficacy as a
single agent, it may hold promise when used as part of a combination therapy.
Thus, it is important to understand the mechanism of ATO action to identify
targets to develop rational combination therapies to increase its therapeutic
spectrum. Indeed, using this strategy, we identified one potential combination
therapy involving arsenic and the vitamin E-derivative trolox, which enhances
tumor killing while protecting against toxicity.135,183
Our laboratory and others have described a pathway required for ATO-induced
apoptosis in APL cells. This pathway involves the generation of reactive oxygen
species (ROS), and the subsequent induction of a specific mitogen-activated
39
protein kinase (MAPK) cascade that includes both stress-activated protein kinase
(SAPK)/ERK kinase 1 (SEK1) and c-Jun N-terminal
kinases
(JNK)
activation.97,148,184 Furthermore, ATO inhibits the pro-survival signals of AKT, a
serine/threonine protein kinase, which negatively regulates the SEK-JNK pathway
on several levels.165 However, little is known about what links ROS production
and activation of the MAPK cascade.
ASK1 is a ubiquitously expressed MAPKKK that activates both JNK and p38
signaling pathways and is a strong mediator of apoptotic signals.167 It
phosphorylates and activates downstream MAPKKs including SEK1 and SEK2 in
response to cellular stress such as oxidative stress, endoplasmic reticulum stress,
calcium overload and receptor-mediated inflammatory signals.168-170 Cellular
stress has been associated with many cancers and may be responsible for
sensitivity to certain drugs and contribute to regulation of cellular proliferation,
DNA damage and genomic instability.113 Thus, ASK1 plays a vital role in limiting
the adverse consequences of redox imbalance.
Upstream of SEK1, there are several MAP3K that are implicated in the induction
of the SEK/JNK pathway. However, since stress-induced signaling is strongly
implicated in ASK1 activation, we hypothesized that ASK1 mediates ATOinduced stress signaling and apoptosis. Using MEF cells derived from ASK1-/mice, we provide evidence that ASK1 was necessary for ATO-induced apoptosis
and JNK activation. We then investigated the role of ASK1 in NB4 cells and
demonstrated a dose-dependent and time-dependent increase in ASK1 activation
following treatment with ATO. However, knockdown of ASK1 in NB4 cells
enhanced susceptibility to ATO.
The same phenotype was observed in the
knockdown of SEK1 in NB4 cells and the knockdown of ASK1 in MCF-7 breast
cancer cells. To determine whether the opposite actions of ASK1 on ATOinduced apoptosis were an inherent difference between normal and malignant
cells, we transformed ASK1-/- MEFs with E1A and RAS oncogenes.
Transformation significantly increased sensitivity to ATO-induced apoptosis and
40
growth inhibition, suggesting that ASK1 can have both pro-apoptotic and antiapoptotic roles depending on transformation state of the cells.
41
Materials and Methods
Cell culture
All cells were grown in a humidified chamber at 37oC in a 5 % CO2 environment.
NB4 cells (kindly provided by Dr M Lanotte)185 and SEK1 knockdown NB4 cells
were maintained in RPMI 1640 media (Wisent) supplemented with 10% fetal
bovine serum (FBS; Wisent) and penicillin/streptomycin (Wisent).
SEK1
knockdown NB4 cells were also maintained in 1 μg/mL puromycin (GIBCO).
MCF-7 (ATCC), 293T (ATCC),wild-type MEF, ASK1-/- MEF cells (kindly
provided by Dr H Ichijo)174 and E1A-Ras transformed ASK1-/- MEF cells were
cultured in Dulbecco’s modified eagle’s medium media (Wisent) supplemented
with 10% FBS and penicillin/streptomycin. E1A-Ras transformed ASK1-/- MEF
cells were also supplemented with 2 μg/mL puromycin.
Trypan Blue Exclusion
NB4 and SEK1 knockdown NB4 cells were seeded at 1 x 105 cells/mL in 24 well
plates. Cells were treated with 0.5 μM, 1 μM, or 2 μM ATO for 2 days. MCF-7,
wild-type MEF, ASK1-/- MEF and E1A-Ras transformed ASK1-/- MEF cells were
seeded at 1.5 x 104 cells/mL in 6 well plates. Cells were treated with 1 μM, 2.5
μM, 5 μM, and 10 μM ATO for 2 days. Viable cells were counted by trypan blue
exclusion (Invitrogen) with a hemacytometer.
CellTiter-Glo Luminescent Cell Viability Assay
Wild-type MEF and ASK1-/- MEF cells were seeded at 1000 cells / 100 μL and
treated with various concentrations of ATO for up to 7 days. Cell viability of
MEF cells were measured by luminescence detection using CellTiter-Glo
Luminescent Cell Viability Assay kit (Promega) as per manufacturer’s
instructions.
42
Sulforhodamine B Colorimetric Assay
Cell viability of wild-type MEF, ASK1-/- MEF, E1A-Ras transformed ASK1-/MEF and MCF-7 cells were measured by absorbance detection using
Sulforhodamine B colorimetric assay. Briefly, cells growing in log phase were
seeded at 500 cells/100 uL in a 96 well plate on day 0. After 24h hours, cells
were treated with 1 μM, 2.5. μM, 5 μM and 10 μM for 3, 5, and 7 days. Cells
were re-treated on day 4. After desired treatment time, the supernatant from the
96 well was removed and the cells were fixed with 10% TCA for 30 minutes at 4
o
C. Following fixation, cells were washed 4 times using alternating dispensing
and removal procedures with distilled water and air-dried overnight at room
temperature.
The 96 well plates were stained with 100 μL of 0.4%
Sulforhodamine B for 30 min with gentle shaking. Following staining, plates
were washed 4 times with 1% acetic acid and air-dried overnight. The following
day, 100 µL of 10 mM Tris base solution was added to each well and absorbance
was measured at 570 nm and 492 nm.
PI stain
NB4 and SEK1 knockdown NB4 cells were seeded at 1 x 105 cells/mL in 24 well
plates. Cells were treated with 0.5 μM, 1 μM, or 2 μM ATO for 2 days. MCF-7,
wild-type MEF, ASK1-/- MEF and E1A-Ras transformed ASK1-/- MEF cells were
seeded at 1.5 x 104 cells/mL in 6 well plates and treated the following day with 1
μM, 2.5 μM, 5 μM or 10 μM ATO for up to 48 hours. Cells were collected and
washed in PBS supplemented with 5% FBS and 0.01 M NaN3. They were then
pelleted and resuspended in 0.2 mL hypotonic fluorochrome solution (50 mg/ml
propidium iodide (Sigma), 0.1% sodium citrate, 0.1% Triton X-100).
Fluorescence was detected by flow cytometry and analysis was performed using
FloJo v7.6.2. Ten thousand events were recorded per sample and analyzed for
cell cycle and apoptosis. Cells undergoing DNA fragmentation and apoptosis
43
were defined as events with fluorescence weaker than the G0-G1 cell cycle peak
(Sub-G0).
JNK Kinase Assay
Wild-type MEF and ASK1-/- MEF cells were seeded at 1 x 108 cells on a 10 cm
plate in 10 mL serum free media for 24 hours. They were then treated with
various concentrations of ATO for 24 hours. Following treatment, cells were
washed once with cold phosphate-buffered saline (PBS) and lysed in the presence
of phosphatase and protease inhibitors.
An anti-JNK1 antibody (Santa Cruz
Biotechnology) and Protein A-Sepharose beads (Sigma) were used to precipitate
JNK1 from 200 μg of cell lysates for each sample by nutation at 4 oC for 1 hour.
Following multiple, successive washes with kinase buffer containing 3 M urea
and then kinase buffer alone, immune complexes were incubated with
glutathione-S-transferase (GST)-c-cjun and [γ32P]ATP for 30 minutes at 30 oC.
GST-c-jun was electrophoresed on an acrylamide gel and visualized by
autoradiography.
Western blotting was performed on samples prior to the
immunoprecipitation to ensure that ATO treatment did not affect absolute JNK1
expression levels.
Western Blot
Cell extracts were prepared by washing 5 x 106 cells with cold PBS and
resuspending cell pellets in 0.3 mL radioimmunoprecipitation assay buffer (50
mmol/L Tris-HCl (pH 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS)
containing protease/phosphatase inhibitors (Roche) at 4 oC.
Extracts were
sonicated using a Sonic Dismembrator (Model 300; Fisher Scientific) for 2 x 10
seconds at 15 % intensity and then centrifuged at 13 000 rpm in a microcentrifuge
at 4 oC for 10 minutes. Supernatants were transferred to fresh tubes and protein
concentrations were determined with the Bio-Rad protein assay (Bio-Rad). To
detect JNK1, E1A, SEK1 and GAPDH, 50 μg of protein was prepared. To detect
44
ASK1, phospho ASK1 T838, and ɑ-actinin, 100 μg of protein was prepared.
Volumes of samples were added to an equal volume of 2x sample buffer and run
on a sodium dodecyl sulfate (SDS)-polyacrylamide gel. Proteins were transferred
to a nitrocellulose membrane (Bio-Rad) and stained with 0.1 % Ponceau S in 5%
acetic acid to ensure equal protein loading. Membranes were then blocked with
5% milk (JNK1, SEK1, E1A, GAPDH and ɑ-actinin) or 5% bovine serum
albumin (ASK1 and phospho ASK1 T838) in TBS containing 0.1% Tween 20
(TBST) for 1 hour at room temperature. The membrane was then hybridized
overnight at 4 oC with antibody against JNK1 (Santa Cruz, 1:500), SEK1 (Cell
Signaling, 1:1000), E1A (Calbiochem, 1:100), GAPDH (Cell Signaling, 1:1000
dilution), Phospho ASK1 T838 (kind gift from Dr. H Ichijo, 1:2000 dilution),175
ASK1 (Cell Signaling, 1:1000 dilution) and ɑ-actinin (Santa Cruz, 1:2000
dilution). Membranes were washed 4 x with in TBST for 10 minutes and then
incubated with a horseradish peroxidase-conjugated secondary antibody (GE
Healthcare) for 1 hour at room temperature. Membranes were once again washed
4 x with TBST for 10 minutes each and the peroxidase activity was visualized by
enhanced chemiluminescence (ECL; GE Healthcare Biosciences-Amersham) or
chemiluminescent HRP substrate (Millipore Coorporation).
shRNA Lentiviral Particle Transduction
NB4 cells were seeded at 0.5 x 106 cells in 2 mL RPMI 1640 media supplemented
with 10% fetal bovine serum and penicillin/streptomycin in a 12 well plate.
MCF-7 cells were seeded at 5 x 104 cells/ mL in 2 mL Dulbecco’s modified
eagle’s medium media supplemented with 10% FBS and penicillin/streptomycin.
The next day, the culture media was replaced with the same media but
supplemented with 5 μg/mL polybrene (Santa Cruz).
To infect cells, 40 μl of
shRNA lentiviral particles for ASK1 (Santa Cruz), SEK1 (Sigma) and their
respective non-target controls were added dropwise to the culture and incubated
overnight. The cell cultures were sequentially expanded in their respective fresh
culture media without polybrene for 4 days. Following expansion, cell cultures
45
were selected in 1 ug/mL (NB4 cells) and 2 ug/mL (MCF-7 cells) puromycinsupplemented media for one week.
Production of Viral Infecting Particles
293T Phoenix cells were seeded at 5 x 106 cells in a 10 cm dish for 24 hours.
Cells were washed and culture media was replaced with 3 mL chloroquine mix
(12.5 μM chloroquine in 3 mL serum free media) and incubated at 37 oC for 5
minutes. After incubation, 1.5 mL of transfection mixture (20 μg of pLPC or
pLPC-E1A-Ras vector in OPTI-MEM I (Invitrogen)) and 1.5 mL of lipofectamine
mixture (80 μL Lipofectamine (Invitrogen) in OPTI-MEM I) were added to the
cells. The plates were incubated for 5 hours at 37 oC followed by the addition of
4 mL of 10% culture media and further incubated overnight at 37 oC. The next
day, the culture media was replaced with 4 mL of fresh media and incubated for
48 hours. The cell culture supernatant was collected and centrifuged at 1200 rpm
for 5 minutes to pellet unwanted residual cells. The cell- free supernatant was
collected for viral infection.
Transformation of MEFs with E1A-Ras
Wild-type and ASK1-/- MEF cells were seeded at 1.5 x 105 cells on a 10 cm plate
and subjected to 2 rounds of viral infection.
Each round consists of first
replacing the culture media with viral supernatant mixture (2 mL viral supernatant
+ 3 mL of culture media supplemented with 2 μg/mL polybrene (Santa Cruz) and
incubating overnight at 37 oC. After the second round of infection, the cells were
incubated in culture media for 24 hours followed by selection in 2 μg/mL of
puromycin for 1 week.
46
Statistical analysis
All statistical tests aiming at evaluating significance were determined by analysis
of variance (one-way analysis of variance) followed by Newman–Keuls post-tests
using Prism version 4.03 (GraphPad software, San Diego, CA, USA).
47
Results
ASK1 is responsible for ATO-induced JNK activation and apoptosis in mouse
embryonic fibroblasts
We have previously shown activation of JNK to be necessary for maximum
induction of apoptosis with ATO treatment.97 Pharmacologic inhibition of JNK in
NB4 cells and genetic inhibition in mouse embryonic fibroblasts (MEFs) deficient
in the direct upstream JNK activator SEK1 showed reduced sensitivity to ATOinduced apoptosis. Given that ASK1 is the direct upstream activator of SEK1, we
first examined whether ASK1 was responsible for ATO-induced apoptosis by
using MEFs derived from ASK1 knockout (ASK1-/-) mice. Wild-type and ASK1/-
MEFs were cultured with a range of concentrations of ATO for 1 week and cell
viability (Figure 1A) was assessed using Titer-Glo assay.
The number of
metabolically active cells were measured and revealed that ATO-induced
cytotoxicity was significantly reduced in ASK1-/- MEFs, with nearly 45% in
reduction in cell viability with the 2.5 µM treatment. Viable cell numbers (Figure
1B) and cell death (Figure 1C) were assessed after a 48 hour treatment with a
variety of ATO concentrations, demonstrating ASK1-/- MEFs to be more resistant
to ATO-induced growth inhibition and apoptosis.
Given that JNK contributes to induction of apoptosis by ATO and the upstream
ASK1 is also responsible for conferring sensitivity to ATO, we hypothesized that
ASK1 plays a role in ATO-induced JNK activation. We expect ASK1 -/- MEFs to
have abrogated ATO-induced JNK activation. Indeed when we examined JNK
activity in response to ATO treatment using a JNK kinase assay, we saw
significant JNK activation in the wild-type MEFs in a dose dependent manner; a
phenomenon not observed in ASK1-/- MEFs (Figure 2). In fact, JNK activation in
the ASK1-/- MEFs was close to baseline untreated control, even following the 5
µM treatment, suggesting a predominant role for ASK1 in ATO-induced stress
signaling in MEFs. The results were surprising considering that deletion of a
single MAP3K, among many, elicited such a prominent effect in ATO signaling.
48
ASK1 is activated by ATO in APL Cells
Given that ASK1 is a MAP3K that plays a predominant role in ATO-induced cell
death in MEFs, we wanted to expand our findings in the prototypical cancer
setting for ATO and explore the role of ASK1 in the APL cell line, NB4. In order
to examine the effect of ATO in ASK1 activation in NB4 cells, an antibody
recognizing the activating phosphorylation site of ASK1 on threonine 838 was
used.
In figure 3A, we show a dose-dependent increase in ASK1
phosphorylation after a 24 hour treatment with a range of ATO concentrations.
Next, we examined the time dependency of ASK1 phosphorylation. When NB4
cells were treated with 2 μM ATO for up to 24 hours, a time-dependent increase
in ASK1 phosphorylation was observed as early as 6 hours and was sustained for
at least 24 hours as illustrated in Figure 3B. These results indicate that ASK1 is
activated by ATO in APL cells.
Knockdown of ASK1 enhances ATO-induced apoptosis in APL cells
To investigate whether ASK1 is necessary in ATO-induced apoptosis in APL
cells, we knocked down ASK1 expression using shRNA lentiviral particles.
Figure 4A confirms the protein expression level of ASK1 is significantly
decreased one week post-puromycin selection. We compared control shRNA and
ASK1 shRNA lentivirus-infected with wild-type NB4 cells for their sensitivity to
0.5- 2 μM ATO. Cell viability was assessed by trypan blue exclusion and cell
death by propidium iodide staining. Unexpectedly, the knockdown of ASK1
significantly enhanced ATO-induced growth inhibition (Figure 4B) and apoptosis
(Figure 4C) compared to control shRNA infected cells and their wild-type
counterparts. These results suggest an anti-apoptotic and pro-survival role for
ASK1 in APL cells.
49
SEK1 knockdown APL cells exhibit enhanced sensitivity to ATO-induced
apoptosis
Previous published work in our lab implicated SEK1 as an important mediator of
ATO-induced JNK activation and apoptosis. SEK1-/- MEFs exhibited enhanced
resistance to ATO-induced apoptosis, similar to the phenotype observed in ASK1/-
MEFs. Given that knockdown of ASK1 in NB4 cells potentiated ATO-induced
apoptosis in contrast to the effects seen in ASK1-/- MEFs, we wanted to determine
the impact of ATO in SEK1 knockdowns in NB4 cells. The genetic approach of
the knockdown made use of SEK1 shRNA lentiviral particles and subsequent
selection in 1 µg/mL puromycin.
The efficiency of the knockdown was
confirmed by the disappearance of SEK1 expression as shown in Figure 5A. A
growth assay revealed that SEK1 knockdown NB4 cells were much more
sensitive to growth inhibition compared to their WT or control shRNA transfected
counterparts (Figure 5B). PI staining also confirmed SEK1 knockdown NB4 cells
to be significantly more sensitive to cell death following ATO treatment (Figure
5C). Taken together, the results support the pro-survival role of ASK1 signaling
in APL cells in response to ATO.
Knockdown of ASK1 enhances ATO-induced apoptosis in MCF-7 cells
The unprecedented negative regulatory role of ASK1 in ATO-induced apoptosis
in APL cells prompted us to further examine the role of ASK1 in another human
cancer cell line to see if the results can be extended into another setting.
Therefore, the MCF-7 human breast cancer cell line, an excellent candidate that
readily undergoes genetic manipulation, was selected for comparison. ASK1 was
knocked down in MCF-7 cells using shRNA lentiviral particle infection and
ASK1 expression was monitored by immunoblot as shown in Figure 6A. We
treated these cells with up to 10 μM ATO due to their reduced sensitivity to ATO
compared to APL cells. After 7 days, cell viability was assessed by SRB assay,
showing enhanced dose-dependent growth inhibition in the ASK1 knockdown
50
MCF-7 cells compared to the control shRNA transfected and wild-type
counterparts as shown in Figure 6B. Correspondingly, the shASK1 MCF-7 cells
also showed enhanced sensitivity to ATO-induced cell death following a 24 hour
treatment (Figure 6C). Taken together, these results suggest that ASK1 may play
an anti-apoptotic and protective role in malignant cell lines.
Transformation of wild-type and ASK1-/- MEFs with E1A-Ras enhances
sensitivity to ATO-induced apoptosis
We next tested whether transformation changed the function of ASK1 by
transforming both ASK1+/+ and ASK1-/- MEFs with E1A-Ras. Ras is a frequently
mutated oncogene in cancers and cooperates with the adenoviral E1A oncoprotein
to transform primary human cells.186 In transformed ASK1+/+ MEFs, expression
of the oncogene was confirmed in an immunoblot for E1A as shown in Figure 7A.
A growth assay and cell death assay revealed the E1A-Ras transformed ASK1+/+
MEFs to be significantly more sensitive to ATO-induced growth inhibition
(Figure 7B) and apoptosis (Figure 7C). These results are consistent with previous
reports that immortalized cells are more sensitive to ATO-induced cell death.187189
Furthermore, we generated E1A-Ras transformed ASK1-/- MEFs.
The
expression level of the E1A-Ras oncoprotein in the transformed cells was
confirmed in Figure 8A by immunoblotting for E1A. When treated with ATO,
the transformed ASK1-/- cells are significantly more sensitive to ATO-induced
growth inhibition (Figure 8B) and apoptosis (Figure 8C) compared to the parental
and empty vector transfected ASK1-/- MEFs, comparable to that of the ASK1+/+
MEFs. These results support the working hypothesis that ASK1 has contrasting
roles in normal versus malignant cells.
51
A
B
C
Figure 1: Deficiency of ASK1 in MEF cells enhances resistance to ATOInduced apoptosis. (A) MEFs from wild-type (WT) mice and ASK1 deficient
(ASK1-/-) mice were cultured in various concentrations of ATO for 7 days. Cell
viability was assessed by Titer-Glo Assay as per manufacturer’s instructions. WT
and ASK1-/- MEFs were also treated with up to 5μM ATO for 48 hours and viable
cell numbers (B) and cell death (C) were assessed by trypan blue exclusion and
PI-staining, respectively. Each bar represents an average of three independent
samples with standard deviation bars shown. Asterisks indicate significant
differences between WT and ASK1-/- MEF treatment matched pairs (* p<0.05; **
p<0.01; *** p<0.001).
52
Figure 2: ASK1 mediates ATO-induced JNK activation in MEFs. WT and
ASK1-/- MEFs were treated for 16 hours with different concentrations of ATO.
Whole cell lysates were used in a JNK kinase assays (top panel) to measure JNK
activity. JNK or β-actin immunoblots (middle and bottom panels respectively)
serve as controls.
53
A
B
Figure 3: ATO activates ASK1 in APL cells. NB4 cells were cultured in media
with various concentrations of ATO for 24 hours (A) or in 2μM ATO for different
durations (B) and immunoblotted for phospho-ASK1-T838, ASK1 and ɑ-actinin.
54
A
B
C
Figure 4: Knockdown of ASK1 in APL cells enhances sensitivity to ATO.
NB4 cells were infected with 200 000 infectious units of virus of ASK1 shRNA
or control shRNA lentiviral particles. Following a 7 day selection in puromycin,
whole cell lysates were prepared and immunoblotted for ASK1 and ɑ-actinin (A).
Transfected cells were also treated with various concentrations of ATO for 48
hours and cell viability was evaluated using trypan blue staining (B). Cell death
was detected by PI Staining (C) following a 24 hour treatment with various
concentrations of ATO. Each bar represents an average of three independent
samples with standard deviation bars shown. Asterisks indicate significant
differences between the control shRNA and ASK1 shRNA treatment matched
pairs (* p<0.05; ** p<0.01; *** p<0.001).
55
A
B
C
Figure 5: Knockdown of SEK1 enhances ATO-induced apoptosis in APL
cells. NB4 cells were infected with 200 000 infectious units of SEK1 shRNA or
control shRNA lentiviral particles. Western blots detailing the expression levels
of SEK1 (A) are shown. Transfected cells were also treated with a range of doses
of ATO for 24 hours and cell viability (B) was evaluated using trypan blue
exclusion while cell death (C) was assayed by PI-staining. Asterisks indicate
significant differences between the control shRNA and SEK1 shRNA treatment
matched pairs (* p<0.05; ** p<0.01; *** p<0.001).
56
A
B
C
Figure 6: Knockdown of ASK1 in human breast cancer cells increases
sensitivity to ATO. MCF-7 cells were infected with 200 000 infectious units of
virus of ASK1 shRNA or control shRNA lentiviral particles. Following a 7 day
selection in puromycin, whole cell lysates were prepared and immunoblotted for
ASK1 and ɑ-actinin (A). Transfected cells were also treated for 7 days with a
range of doses of ATO and cell density was measured using a Sulforhodamine-B
(SRB) assay (B). Cell death (C) was assessed by PI-staining following treatment
with ATO at the indicated concentrations for 2 days. Each bar represents an
average of three independent samples with standard deviation bars shown.
Asterisks indicate significant differences between the control shRNA and ASK1
shRNA treatment matched pairs (* p<0.05; ** p<0.01; *** p<0.001).
57
A
B
C
Figure 7: Transformation of WT MEFs with E1A-Ras increases sensitivity to
ATO. WT MEFs were transfected with 20 μg of pLPC or pLPC-E1A-Ras.
Following a 7 day selection with puromycin, whole cell lysates were prepared and
immunoblotted for E1A and GAPDH (A). Transfected cells were also treated
with various concentrations of ATO for 48 hours and growth inhibition (B) was
assessed by counting live, trypan blue-negative cells and expressing it as a
percentage of viable cells relative to the untreated control. Apoptosis was
assessed by PI staining (C). Each bar represents an average of three independent
samples with standard deviation bars shown. Asterisks indicate significant
differences between EV and E1A-Ras transformed MEF treatment matched pairs
(* p<0.05; ** p<0.01; *** p<0.001).
58
A
B
C
Figure 8: E1A-Ras transformed ASK1-/- MEFs are more susceptible to ATOinduced apoptosis than their parental counterparts. ASK1-/- MEFs were
transfected with 20 μg of pLPC or pLPC-E1A-Ras. Whole cell lysates were
prepared and immunoblotted for E1A and GAPDH 7 days after selection in 2
μg/mL puromycin (A). Transfected cells were evaluated for cell viability (B)
using SRB assay after culturing the cells in the indicated concentrations of ATO
for 7 days. Transfected cells were also treated with a range of concentrations of
ATO for 48 hours and cell death (C) was assessed using PI-staining. Each bar
represents an average of three independent samples with standard deviation bars
shown. Asterisks indicate significant differences between the EV and E1A-Ras
transformed MEF treatment matched pairs (* p<0.05; ** p<0.01; *** p<0.001).
59
Discussion
Our lab has linked ATO-induced ROS and JNK activation to apoptosis. We have
also identified SEK1, a MAP2K directly upstream of JNK activation, to be
necessary for maximum induction of JNK activation and apoptosis by ATO.97
Located directly upstream of SEK1, ASK1 is one of many MAP3K situated on
the top of a 3 tier evolutionarily conserved signaling cascade, which senses
various internal and external stresses to elicit a wide variety of cellular responses
including cell death and proliferation.190 In this study, we sought to identify the
role of ASK1 in ATO-induced apoptosis.
Using MEF cells derived from ASK1-/- mice, we provide evidence that ASK1
mediates ATO-induced apoptosis and JNK activation. We then investigated the
role of ASK1 in NB4 cells and demonstrated a dose-dependent and timedependent increase in ASK1 activation following treatment with ATO. However,
knockdown of ASK1 in NB4 cells enhanced susceptibility to ATO, undergoing
apoptosis and growth inhibition more than their wild type counterparts. The same
impact was observed in the knockdown of SEK1, a direct downstream MAP2K of
ASK1, resulting in an increase in ATO sensitivity. Furthermore, the increased
susceptibility to ATO was also observed in the human breast cancer cell line
MCF-7 when we knocked down ASK1. This led us to postulate that ASK1 had a
pro-apoptotic function in non-transformed fibroblasts, but was pro-survival in
malignant cells. Indeed, transformation of WT and ASK1-/- MEFs significantly
increased their sensitivity to ATO-induced apoptosis and growth inhibition,
highlighting differences of ASK1 in normal versus transformed cells. Taken
together, these results suggest that the role of ASK1 depends on cellular context,
having opposite roles in normal versus transformed cell types.
To determine whether ASK1 activation is necessary for ATO-induced apoptosis,
our initial experiments examined the effects of ATO on cell growth and cell death
in ASK1-/- MEFs. These experiments revealed that ASK1 played a predominant
role in ATO-induced apoptosis and JNK activation since ASK1-/- MEFs were
60
significantly resistant to ATO-induced apoptosis accompanied by remarkable
reduction of sustained JNK activation compared to wild-type controls.
The
complete abrogation of JNK activity in these cells for at least 24 hours suggests
that ASK1 plays a pivotal role in ATO-induced stress signaling and apoptosis.
The results are surprising, considering the numerous MAP3K found upstream of
JNK activation in stress-induced signaling but are consistent with the documented
role of ASK1.
Since ASK1 has been shown to be necessary in ATO-induced cell death in MEFs,
we wanted to determine if this remained true in a cancer setting.
In NB4 cells,
the activation of ASK1 induced by ATO occurred in a dose-dependent and timedependent manner, correlating with our published results on ROS accumulation,
JNK activation, growth inhibition and cell death. In this regard, ATO appears to
play a positive role in the induction of apoptosis. However, the present study also
suggests that ASK1 may mediate pro-survival signals in APL cells. Our results
from the ASK1 knockdown experiments in NB4 cells showed a markedly
increased sensitivity to ATO-induced cell death and growth inhibition in the
ASK1 knockdowns compared to their parental counterparts. This is consistent
with results from another group, reporting a pro-survival role for ASK1.191 These
results suggest that ASK1 may play a pro-survival role in transformed cells. To
confirm these results, it will be necessary to validate the other signaling
requirements in the ASK1 knockdown NB4 cells and evaluate JNK activity in
response to ATO. It will also be interesting to evaluate p38 activation because
several studies have implicated p38 as a negative regulator in ATO-induced of
apoptosis.123,147
We wanted to determine if the pro-survival role of ASK1 could be reproduced in
a solid tumour-derived cell line, so we expanded our results to the human breast
cancer cell line MCF-7, a relatively easy model to perform genetic manipulations.
Consistent with the results in NB4 cells, ATO induced significantly more
apoptosis and growth inhibition in a dose-dependent and time-dependent manner
61
when ASK1 was knocked down. Taken together, these results suggest that ASK1
mediates a pro-survival role, but requires further validation of its signaling
requirements. While these results are surprising, stress-induced ASK1 activation
has been reported to have other consequences. Expression of constitutively active
ASK1 in the rat pheochromocytoma cell line PC12 was shown to induce neurite
outgrowth, activate expression of mature neuron-specific proteins, and survive in
serum-starved conditions, suggesting that ASK1 may mediate signals for
differentiation and survival.172 ASK1 has also been implicated in G-proteincoupled
receptor
agonist-Induced
NF-κB
activation
in
cardiomyocyte
hypertrophy.171,173 These studies support the notion that ASK1 has a variety of
functions depending on the biological context and is not limited only to a proapoptotic function.
It remains a possibility that the differences in the role of ASK1 in normal cells
and the two different malignant cell lines are attributed to a normal versus
malignant background. To test this hypothesis, ASK1-/- MEFs were transformed
with E1A-Ras.
Compared to their parental counterparts, the E1A-Ras
transformed ASK1-/- MEFs were more sensitive to ATO-induced apoptosis and
growth inhibition suggesting enhanced ATO cytotoxicity in the transformed cells.
These results are comparable to the impact of transforming WT MEFs. The
increased cytotoxicity of ATO in transformed cells probably reflect cellular
changes associated with the transformed state, which can include shortening of
the cell cycle, excision repair deficiency, or an increase in DNA replicon size and
many biochemical changes.192 In addition, ASK1 signaling in response to ATO
seems to play a pro-survival role in transformed cells.
The current results
demonstrating a difference in ATO sensitivity between normal and malignant
cells as well as the role of ASK1 in these settings are important because they shed
light on ATO as a directed drug targeting tumors and sparing normal cells.
The role of ASK1 in normal and malignant cells is not only attributed to ASK1
activation, but to ASK1 downstream signaling. Published work from our lab has
62
demonstrated that genetic knockout of SEK1, a direct downstream target of
ASK1, conferred reduced sensitivity to ATO-induced apoptosis and JNK
activation in MEFs. Here, we show that SEK1 knockdown in NB4 cells resulted
in significantly enhanced sensitivity to ATO, mirroring the effects of the ASK1
knockdown NB4 cells. These results support the notion that ASK1 activation and
ASK1 signaling are responsible for the differences in sensitivity to ATO in the
normal and malignant setting. However, we cannot exclude the possibility of
inherent differences between mouse and human cell lines or a knockdown versus
a genetic knockout. To address the first possibility, it would be interesting to
perform an ASK1 knockdown in a malignant mouse cell line and assess its
sensitivity to ATO.
The latter possibility can be evaluated by an ASK1
knockdown in wild-type MEF cells.
Given that ATO has been reported to activate p38 and JNK signaling
cascade,167,174 one instance where ATO signaling leads to cell survival is due to
the increased activation of p38 relative to JNK. 10,11 Activation of p38 signaling
cascade has been shown to negatively regulate the induction of apoptosis in
leukemic cell lines, where pharmacologic inhibition of p38 potentiates ATOinduced apoptosis and growth inhibition,123 as well as JNK activation.147 It would
be interesting to evaluate and compare p38 activation in our ASK1 knockdown
NB4 cells and ASK1 deficient MEFs in response to ATO. If the activity of the
p38 pathway accounts for ATO sensitivity between normal and malignant cells,
we would expect greater relative p38 activation in the ASK1 deficient MEFs
following ATO treatment. Another possibility for the enhanced sensitivity to
ATO in normal versus malignant cells could be attributed to the differential
expression of ASK2, an important component of the ASK1 signalosome. ASK1
functions as part of two different high-molecular mass complexes to exert its
kinase activity: one with a core structure consisting of a heteromeric complex
with ASK2 and one whose core structure is composed of a homo-oligomer of
ASK1.176,193 Accumulating evidence implicates ASK2 as an important component
of the ASK1 signalosome where ASK1 is required to stabilize ASK2 to allow
63
ASK2 to activate ASK1 by direct phosphorylation to accelerate the
autophosphorylation of ASK1.194 The increased sensitivity to ATO in the ASK1
knockdown NB4 cells may be at least partly attributed to an increase in ASK2
expression as a consequence of the incomplete ASK1 depletion as a compensatory
mechanism. This would imply an increase in the formation of the more efficient
ASK1 heteromeric complex, resulting in increased ATO-induced ASK1 signaling
and cell death.
Overall, our results suggest that ASK1 has an important context-dependent role in
ATO sensitivity. In the context of malignant cells, ASK1 may play a pro-survival
role. Conversely, ASK1 may mediate apoptotic signals in normal cells. These
differences in malignant versus normal cells make ASK1 an interesting
therapeutic target to enhance ATO sensitivity in malignant cells.
64
CHAPTER 3: REGULATION OF ASK1 IN ATO-INDUCED APOPTOSIS
Introduction
ASK1 is a strong mediator of pro-apoptotic signals depending on the cellular
context therefore its activity is highly regulated in the cell. ASK1 has been shown
to be regulated by numerous players and mechanisms.
To date, there are 2 different phosphatases that have been reported to inactivate
ASK1 through the dephosphorylation of the activating phosphorylation site on
ASK1 on threonine 838 (humans) and threonine 845 (mice). Protein phosphatase
5 (PP5) is a serine/threonine protein that binds to ASK1 and dephosphorylates
ASK1 in response to ROS stress thus providing a negative feedback
mechanism.195 In addition, the serine/threonine phosphatase, PP2Cε, negatively
regulates ASK1 activation under basal non-stressed conditions to maintain ASK1
in its dephosphorylated state.196
Another regulator of ASK1 is AKT, a serine/threonine protein kinase that
mediates cell survival signals by the phosphorylation and inactivation of proapoptotic proteins including BAD158, caspase-9159 and forkhead (FKHR) family of
transcription factors.160 AKT has been reported to suppress ASK1 activity induced
by ROS and overexpression of ASK1 in 293 cells by associating with ASK1 and
phosphorylating its serine 83 residue.162
Tumor necrosis factor-ɑ receptor-associated factors (TRAFs) play an essential
role in ASK1 activity regulation. ASK1 has been shown to associate with many
members of the TRAF family including TRAF1, TRAF2, TRAF3, TRAF5 and
TRAF6 but only TRAF2 and TRAF6 play an important role in ASK1 activity,
serving as members of the higher molecular mass complex ASK1 signalosome.197
Noguchi et al. showed that H2O2-induced ASK1 activation and cell death were
markedly reduced in cells from Traf2-/- and Traf6-/- mice, consistent with the role
of TRAF2 and TRAF6 in activating ASK1.198
65
ASK1 is negatively regulated by 14-3-3 proteins, a highly conserved family of
phospho-serine/phospho-threonine binding proteins with a multitude of functions,
including roles in metabolism, protein trafficking, signal transduction, apoptosis
and cell-cycle regulation.199 Under basal non-stressed conditions, 14-3-3 binds to
ASK1 on the basally phosphorylated serine 967 residue on the C-terminal domain
of ASK1 to inhibit it.200 Upon stress-induced signaling, ASK1 interacting protein
I (AIP1), a Ras-GAP protein that forms a complex with ASK1, facilitates the
dissociation of 14-3-3 from ASK1 by recruiting the phosphatase PP2A which
dephosphorylates ASK1 at the serine 967 residue (Figure 3.1).201
The most well-characterized negative regulator of ASK1 is thioredoxin 1 (Trx1),
a ubiquitously expressed protein that has a variety of biological functions
including regulating cellular redox environment, cell proliferation and
apoptosis.202 Trx1 contains two cysteine residues within the redox active center
responsible for its reducing ability.203 Once oxidized, Trx1 can become reduced
by the flavoprotein Trx reductase 1(TrxR1).204 The interaction of Trx1 and ASK1
depends on the redox status of Trx1. Reduced Trx1 binds to ASK1 under basal
non-stressed conditions at the N-terminal region to inhibit ASK1.168 ROS converts
Trx1 to its oxidized form, releasing ASK1 from Trx1 inhibition (Figure 3.1).
Given that ATO induces apoptosis through an ROS-dependent and ASK1dependent mechanism in normal cells, we hypothesize that Trx1 plays an
important in regulating sensitivity to ATO. Modulation of Trx1 redox state is
therefore a potentially attractive strategy to enhance ATO-induced apoptosis.
Recently, TrxR1 has been suggested as a new target for anticancer drug
development.205
Here, we provide preliminary data on elucidating the role of Trx1 as a regulator of
ASK1 in ATO-induced apoptosis. We provide evidence that ATO may activate
ASK1 signaling by disrupting the interaction between ASK1 and Trx1.
66
Furthermore, inhibition of TrxR1, which prevents reduction of Trx1, may enhance
ATO-induced apoptosis.
Figure 3.1 Regulation of ASK1 by Trx and 14-3-3
67
Materials and Methods
Cell culture
All cells were grown in a humidified chamber at 37oC in a 5 % CO2 environment.
NB4 cells (kindly provided by Dr M Lanotte)185 were maintained in RPMI 1640
media (Wisent) supplemented with 10% fetal bovine serum (FBS; Wisent) and
penicillin/streptomycin (Wisent).
Immunoprecipitation
NB4 cells were seeded at 2 x106 cells in a T25 flask and allowed to grow
overnight. They were treated with various concentrations of ATO for 24 hours or
in 2 μM ATO for up to 24 hours. Cells were lysed at 4 oC in lysis buffer (50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton x-100) supplemented with protease
and phosphatase inhibitors (Roche). After determining the protein concentration
by Bradford Assay, 750 μg lysates in 500 μL of lysis buffer were prepared and
pre-cleared in protein A agarose beads (Sigma) for 1 hour at 4 oC under gentle
nutation. An aliquot of 50 μL was taken as the input control for Western blotting
to confirm an equal amount of ASK1 and Trx1. Lysates were nutated in 1 μg of
ASK1 antibody (Santa Cruz) overnight at 4 oC. The cell lysates were then nutated
with 40 μL of 50% beads for 6 hours at 4 oC to capture the immune complexes.
The beads were washed extensively with lysis buffer and the protein was eluted in
55 μL of 2X SDS loading buffer. The proteins were resolved on a SDS-PAGE for
subsequent Western blotting.
Thioredoxin Reductase Assay
NB4 cells were seeded at 2 x106 cells in a T25 flask and allowed to grow
overnight. They were treated with various concentrations of ATO for 24 and
whole cell lysates were prepared in radioimmunoprecipitation assay buffer (50
mmol/L Tris-HCl (pH 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS)
68
containing protease/phosphatase inhibitors (Roche) at 4 oC. The measurement of
TrxR1 activity was detected using a DTNB reduction assay kit from Cayman
Chemical Company following the manufacturer’s instructions. Briefly, 160 ug of
cell lysate in 20 uL was mixed thoroughly with 20 μL NADPH, 20 μL (5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), with or without 20 μL sodium
aurothiomalate (ATM) and a variable volume of TrxR assay buffer (50 mM
potassium phosphate, pH 7.0, containing 50 mM KCl, 1 mM EDTA, and 0.2
mg/mL BSA) to make it up to a volume of 200 μL in a 96 well plate. The
reaction solutions using rat liver TrxR instead of sample cell lysate was set up as a
positive control. The microtiter plate was shaken gently for 10 seconds and the
absorbance was recorded once every minute at 405-414 nm using a plate reader.
The reaction rate of each sample was expressed as a percentage of the untreated
sample.
PI stain
NB4 were seeded at 1 x 105 cells/mL in 24 well plates. Cells were treated with 1
μM ATO in combination with 1 or 2.5 μM Auranofin for 24 hours. Cells were
collected and washed in PBS supplemented with 5% FBS and 0.01 M NaN3.
They were then pelleted and resuspended in 0.2 mL hypotonic fluorochrome
solution (50 mg/ml propidium iodide (Sigma), 0.1% sodium citrate, 0.1% Triton
X-100).
Fluorescence was detected by flow cytometry and analysis was
performed using FloJo v7.6.2. Ten thousand events were recorded per sample and
analyzed for cell cycle and apoptosis. Cells undergoing DNA fragmentation and
apoptosis were defined as events with fluorescence weaker than the G0-G1 cell
cycle peak (Sub-G0).
Western Blot
Cell extracts were prepared by washing 5 x 106 cells with cold PBS and
resuspending cell pellets in 0.3 mL radioimmunoprecipitation assay buffer (50
69
mmol/L Tris-HCl (pH 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS)
containing protease/phosphatase inhibitors (Roche) at 4 oC.
Extracts were
sonicated using a Sonic Dismembrator (Model 300; Fisher Scientific) for 2 x 10
seconds at 15 % intensity and then centrifuged at 13 000 rpm in a microcentrifuge
at 4 oC for 10 minutes. Supernatants were transferred to fresh tubes and protein
concentrations were determined with the Bio-Rad protein assay (Bio-Rad). To
detect phospho-14-3-3 serine 58 and 14-3-3, 50 μg of protein was prepared.
Volumes of samples were added to an equal volume of 2x sample buffer and run
on a sodium dodecyl sulfate (SDS)-polyacrylamide gel. Proteins were transferred
to a nitrocellulose membrane (Bio-Rad) and stained with 0.1 % Ponceau S in 5 %
acetic acid to ensure equal protein loading. Membranes were then blocked with
5% milk in TBS containing 0.1% Tween 20 (TBST) for 1 hour at room
temperature. The membrane was then hybridized overnight at 4 oC with antibody
against phospho-14-3-3 serine 58 (Abcam, 1:1000), 14-3-3 (Abcam, 1:1000), JNK
(Santa Cruz, 1:500) and Trx1 (Santa Cruz, 1:500). Membranes were washed 4 x
with TBST for 10 minutes each wash and then incubated with a horseradish
peroxidase-conjugated secondary antibody (GE Healthcare) for 1 hour at room
temperature. Membranes were once again washed 4 x with TBST for 10 minutes
each and the peroxidase activity was visualized by enhanced chemiluminescence
(ECL; GE Healthcare Biosciences-Amersham).
Statistical analysis
All statistical tests aiming at evaluating significance were determined by analysis
of variance (one-way analysis of variance) followed by Newman–Keuls post-tests
using Prism version 4.03 (GraphPad software, San Diego, CA, USA).
70
Results
ATO disrupts the interaction between ASK1 and Trx1
One model of ASK1 regulation stipulates that ASK1 is held in an inactive form
by reduced Trx1, but becomes liberated when Trx1 is oxidized. To determine if
ATO affects the interaction between ASK1 and Trx1, NB4 cells were treated with
various concentrations of ATO for 24 hours (Figure 1A) or in 2 μM ATO for up
to 24 hours (Figure 1B). The cell lysates were immunoprecipitated for ASK1 and
immunblotted for Trx1. The results indicate a strong basal association between
ASK1 and Trx1 that is lost with as little as 0.5 μM ATO in 24 hours or 2 μM
ATO in 1 hour. The results suggest the ATO disrupts the interaction between
ASK1 and Trx1.
Inhibition of thioredoxin reductase activity by ATO
Thioredoxin reductase converts oxidized thioredoxin back into reduced
thioredoxin. We wanted to determine whether ATO can inhibit TrxR1 activity,
resulting in oxidized Trx1. NB4 cells were cultured in various concentrations of
ATO for 24 hours and TrxR1 activity was assessed by a DTNB reduction assay
from Cayman Chemical Company. TrxR1 activity was significantly reduced with
as little as 0.5 μM ATO. When the ATO concentration was greater than 1 μM
ATO, TrxR1 activity was completely inhibited. These results suggest that ATO
may mediate ASK1 signaling partly through preventing the restoration of reduced
thioredoxin.
Inhibition of TrxR1 enhances ATO-induced apoptosis
Given the role of reduced thioredoxin and its restoration by TrxR1 to inhibit
ASK1 signaling, we hypothesized that inhibition of TrxR1 activity may enhance
ATO-induced apoptosis. To test this, we treated NB4 cells with both ATO and an
Auranofin, a TrxR1 inhibitor.
We assessed cell death in response to the
71
combination treatment and observed significantly enhanced cell death with
combination treatment compared to ATO alone. These results are promising,
indicating a potential combination therapy with ATO and thioredoxin reductase
inhibitors.
ATO induces serine 58 phosphorylation of 14-3-3 proteins
Independent of the Trx1 system of ASK1 regulation, 14-3-3 may also sequester
ASK1 by direct binding as a dimer. Since the serine 58 phosphorylation on 14-33 is required for dimerization and client protein binding,206 we wanted to
determine if ATO signaling phosphorylates 14-3-3 at serine 58 thereby preventing
dimerization and interaction with ASK1. Indeed, when we evaluated phospho-143-3 serine 58 protein levels with immuoblotting in NB4 cells treated with ATO,
we observed a time-dependent increase in serine 58 phosphorylation. This is
consistent with the 14-3-3 model of ASK1 regulation.
72
A
B
Figure 1: ATO disrupts interaction between ASK1 and Trx1. NB4 cells were
cultured in various concentrations of ATO for 24 hours (A) or in 2 μM ATO for
different durations (B). The cell lysates were immunoprecipitated for ASK1 and
the complexes were immunoblotted for Trx1.
73
Figure 2: ATO decreases thioredoxin reductase activity. NB4 cells were
treated with 0.5, 1 and 2 μM ATO for 24 hours. Thioredoxin reductase activity
was assessed using the Thioredoxin Reductase Assay Kit (a DTNB reduction
assay) from Cayman Chemical Company.
Asterisks indicate significant
differences between the untreated control and the indicated treatment (* p<0.05;
** p<0.01; *** p<0.001).
74
Figure 3: A thioredoxin reductase inhibitor enhances ATO-induced
apoptosis. NB4 cells were treated with 1 μM ATO and various concentrations of
Auranofin, a thioredoxin reductase inhibitor. Apoptosis was assessed by PI
staining. Asterisks indicate significant differences between the ATO treated and
the combination treatment (* p<0.05; ** p<0.01; *** p<0.001).
75
Figure 4: ATO induces serine 58 phosphorylation of 14-3-3. NB4 cells were
treated with 1 μM ATO for up to 6 hours and immunoblotted for phospho-14-3-3S58 and 14-3-3.
76
Discussion
The role of ASK1 in ATO-induced apoptosis depends on cellular context. It is
capable of playing both a pro-apoptotic and pro-survival role; therefore its
regulation can determine the sensitivity to ATO.
However, the molecular
mechanism of ASK1 regulation in response to ATO is not well known.
Here we explored the role of Trx1 in the regulation of ASK1 in ATO-induced
apoptosis. Reduced Trx1 has been reported to bind and sequester ASK1, while
oxidized Trx1 dissociates from ASK1.168 Since ATO induces apoptosis through
strong oxidative stress signals, the oxidation of Trx1 may be induced by ATO
which leads to ASK1 activation and ASK1-mediated cell death. Our preliminary
results indicate that ATO disrupts the interaction between ASK1 and Trx1 and
inhibits the activity of thioredoxin reductase 1, the enzyme that converts oxidized
Trx1 back into reduced Trx1. We further show that a thioredoxin reductase
inhibitor may enhance ATO-induced cytotoxicity. Independent from the Trx1
regulation of ASK1, we also provide preliminary evidence that ATO induces the
phosphorylation of 14-3-3 at serine 58, thereby preventing the dimerization of 143-3 and the sequestration of ASK1.
Consistent with the working hypothesis, ATO treatment in NB4 cells disrupted
the interaction between ASK1 and Trx1 in a sustained manner, with as little as 0.5
μM, a concentration that does not induce apoptosis. Temporally, the ASK1-Trx1
interaction disruption preceded ATO-induced cell death and correlates with ATOinduced ROS generation. Presumably, reduced Trx1 is oxidized to release ASK1
thus we expect the ratio of oxidized to reduced Trx1 to increase. To test this
hypothesis, it would be interesting to evaluate the change in redox state induced
by ATO.
Whole cell lysates treated with ATO can by alkylated with 4-
acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) and run on a nonreducing Western blot to differentiate between the reduced (alkylated) and nonreduced (non-alkylated) proteins. We also expect that co-treatment with ATO and
77
N-acetyl-cysteine (NAC) at concentrations that decrease ATO-signaling will
restore the oxidized Trx1:reduced Trx1 ratio.
The sustained loss in interaction between ASK1 and Trx1 in response to ATO
could be partly attributed to the ATO-induced decrease in TrxR1 activity. While
0.5 μM ATO significantly decreased TrxR1 activity, a complete loss of TrxR1
activity was observed with the 1 μM treatment, the minimum dose needed to
induce apoptosis in NB4 cells.
To confirm the role of TrxR1, it would be
interesting to overexpress TrxR1 and assess the impact on ATO-induced JNK
activation and ASK1 activation.
We have begun to identify inhibitors of Trx1 and TrxR1 to maintain Trx1 in its
oxidized state and unable to bind ASK1 thus synergizing with ATO cytotoxicity.
One such compound is Auranofin, a potent thioredoxin reductase inhibitor.207
Preclinical studies have already implicated Auranofin in inducing apoptosis in a
variety of cancer lines including cisplatin-resistant human ovarian cancer cells208
and adriamycin-resistant human K562 chronic myeloid leukemia cells209 through
the inhibition of thioredoxin reductase.
Our data suggests that Auranofin
enhances ATO-induced apoptosis in NB4 cells and that targeting TrxR1 is a
potential basis for cancer therapy by ATO. However, Auranofin may not mediate
its pro-apoptotic effects through ASK1 regulation. We have previously shown
that ASK1 play opposite roles in transformed versus normal cells. While it is
activated in NB4 cells in response to ATO, it may mediate pro-survival signals
(Chapter 2). Since inhibition of TrxR1 with Auranofin results in more oxidized
Trx1 and ASK1 activation, we would expect increased cell survival in NB4 cells
instead of the observed increase in cell death. Thus, inhibition of TrxR1 may
induce cell death through an ASK1-independent mechanism. It is possible that
Auranofin may enhance ATO-induced cytotoxicity independent of Trx1
regulation since it has other targets.
78
Independent of the role we find for Trx1 in ASK1 regulation, other proteins may
also regulate ASK1 activation in response to ATO although their contribution and
exact mechanism remains to be elucidated.
These proteins include 14-3-3
proteins. Our data shows that serine 58 of 14-3-3 becomes phosphorylated in
response to ATO, suggesting that 14-3-3 dimerization and protein binding is
inhibited. While the release of ASK1 is one possibility of integrating apoptotic
signals, 14-3-3 is also implicated in promoting survival by sequestering proapoptotic
proteins
upon
phosphorylation
by
AKT.210
ATO-induced
phosphorylation of the serine 58 residue of 14-3-3 may also prevent 14-3-3 from
sequestering pro-apoptotic proteins, therefore leading to the release of proapoptotic proteins and cell death.
In summary, we provide preliminary data that Trx1 plays an important role in the
regulation of ASK1 and that targeting TrxR1 is a potential basis for cancer
therapy by ATO.
The use of ATO and Auranofin is a potential rational
combination therapy that increases the cytotoxicity of ATO and may increase its
therapeutic spectrum.
79
CHAPTER 4: CONCLUSION
ATO has been demonstrated to possess important anti-tumour properties and is an
effective treatment for APL. It has become increasingly clear through numerous
clinical studies that elucidating the mechanism of ATO-induced apoptosis will
allow for the identification of targets to generate rational combination therapies,
enhance the cytotoxicity of ATO, and increase its therapeutic spectrum. Our lab
has already elucidated a potential pathway involving ROS generation and JNK
activation. Our findings in Chapter 2 aimed to characterize the role of ASK1, a
well-documented mediator of stress-induced apoptosis, in our proposed pathway
of ATO-induced cell death. Interestingly, we found opposite roles of ASK1 in
normal versus transformed cells. In normal un-transformed cells, ASK1 plays a
pro-apoptotic role. Transformation significantly increased sensitivity to ATOinduced apoptosis and growth inhibition, suggesting that ASK1 can have both
pro-apoptotic and anti-apoptotic roles depending on the transformation state of the
cells.
The importance of ASK1 in sensitivity to ATO led us to investigate the regulation
of ASK1. We focused on the role of Trx1 since it is the most well characterized
regulator of ASK1. Our preliminary data shows that ATO disrupts the interaction
between ASK1 and Trx1. Furthermore, it decreases the activity of TrxR1,
presumably leading to more oxidized Trx and enhanced ASK1 signaling.
Interestingly, co-treatment of NB4 cells with ATO and Auranofin, an inhibitor of
TrxR1, resulted in enhanced cell death. However, inhibition of TrxR1 may
increase cell death in an ASK1-independent mechanism since ASK1 was shown
to play a pro-survival role in NB4 cells. While the mechanism of action remains
to be elucidated, targeting TrxR1 is a potential basis for cancer therapy by ATO.
80
REFERENCES
1
Institute, N. C. What is Cancer?,
<http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer>
(2012).
2
Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next
generation. Cell 144, 646-674, doi:10.1016/j.cell.2011.02.013 (2011).
3
Statistics, C. C. S. s. S. C. o. C.
(ed Canadian Cancer Society)
(Canadian Cancer Society Toronto, 2012).
4
Anand, P. et al. Cancer is a Preventable Disease that Requires Major
Lifestyle Changes. Pharmaceutical Research 25, 2097-2116,
doi:10.1007/s11095-008-9661-9 (2008).
5
NIH. Vol. 06-1566 (ed National Cancer Institute) 71 (2005).
6
Hejmadi, M.
7
MedicineNet.com. Leukemia Symptoms, Causes, Treatment,
46 (Momna Hejmadi & Ventus Publishing ApS, 2010).
<http://www.medicinenet.com/leukemia/page6.htm> (2012).
8
Bennett, J. M. et al. Proposals for the classification of the acute
leukaemias. French-American-British (FAB) co-operative group. Br J
Haematol 33, 451-458 (1976).
9
Wang, Z.-Y. & Chen, Z. Acute promyelocytic leukemia: from highly
fatal to highly curable. Blood 111, 2505-2515, doi:10.1182/blood2007-07-102798 (2008).
10
Liu, J.-X., Zhou, G.-B., Chen, S.-J. & Chen, Z. Arsenic compounds:
revived ancient remedies in the fight against human malignancies.
Current Opinion in Chemical Biology 16, 92-98,
doi:10.1016/j.cbpa.2012.01.015 (2012).
11
de The, H., Chomienne, C., Lanotte, M., Degos, L. & Dejean, A. The
t(15;17) translocation of acute promyelocytic leukaemia fuses the
retinoic acid receptor [alpha] gene to a novel transcribed locus. Nature
347, 558-561 (1990).
81
12
Melnick, A. & Licht, J. D. Deconstructing a Disease: RAR{alpha}, Its
Fusion Partners, and Their Roles in the Pathogenesis of Acute
Promyelocytic Leukemia. Blood 93, 3167-3215 (1999).
13
Hong, S.-H., Yang, Z. & Privalsky, M. L. Arsenic Trioxide Is a Potent
Inhibitor of the Interaction of SMRT Corepressor with Its
Transcription Factor Partners, Including the PML-Retinoic Acid
Receptor α Oncoprotein Found in Human Acute Promyelocytic
Leukemia. Molecular and Cellular Biology 21, 7172-7182,
doi:10.1128/mcb.21.21.7172-7182.2001 (2001).
14
Jansen, J. H. & Lowenberg, B. Acute promyelocytic leukemia with a
PLZF-RARalpha fusion protein. Semin Hematol 38, 37-41 (2001).
15
Redner, R. L., Rush, E. A., Faas, S., Rudert, W. A. & Corey, S. J. The
t(5;17) variant of acute promyelocytic leukemia expresses a
nucleophosmin-retinoic acid receptor fusion. Blood 87, 882-886
(1996).
16
Wells, R. A., Catzavelos, C. & Kamel-Reid, S. Fusion of retinoic acid
receptor alpha to NuMA, the nuclear mitotic apparatus protein, by a
variant translocation in acute promyelocytic leukaemia. Nat Genet 17,
109-113, doi:10.1038/ng0997-109 (1997).
17
Cunningham, I. et al. Acute promyelocytic leukemia: treatment results
during a decade at Memorial Hospital. Blood 73, 1116-1122 (1989).
18
Huang, M. et al. Use of all-trans retinoic acid in the treatment of acute
promyelocytic leukemia. Blood 72, 567-572 (1988).
19
Castaigne, S. et al. All-trans retinoic acid as a differentiation therapy
for acute promyelocytic leukemia. I. Clinical results [see comments].
Blood 76, 1704-1709 (1990).
20
Shen, Z.-X. et al. Use of Arsenic Trioxide (As2O3 ) in the Treatment
of Acute Promyelocytic Leukemia (APL): II. Clinical Efficacy and
Pharmacokinetics in Relapsed Patients. Blood 89, 3354-3360 (1997).
82
21
Dilda, P. J. & Hogg, P. J. Arsenical-based cancer drugs. Cancer
Treatment Reviews 33, 542-564, doi:10.1016/j.ctrv.2007.05.001
(2007).
22
Garelick, H., Jones, H., Dybowska, A. & Valsami-Jones, E. Vol. 197
Reviews of Environmental Contamination and Toxicology 17-60
(Springer New York, 2008).
23
Cohen, S. M., Arnold, L. L., Eldan, M., Lewis, A. S. & Beck, B. D.
Methylated arsenicals: the implications of metabolism and
carcinogenicity studies in rodents to human risk assessment. Crit Rev
Toxicol 36, 99-133 (2006).
24
Konkola, K. More than a coincidence? The arrival of arsenic and the
disappearance of plaque in early modern Europe. J Hist Med Allied Sci
47, 186-209 (1992).
25
Laboratories, S. R.
26
Group, K. s. G.
27
Webminerals.
28
Smedley, P. L. & Kinniburgh, D. G. United Nations Synthesis Report
on Arsenic in Drinking Water: Chapter1, Source and behaviour of
arsenic in natural waters. (2009).
<http://www.who.int/water_sanitation_health/dwq/arsenicun1.pdf>.
29
Platanias, L. C. Biological Responses to Arsenic Compounds. J Biol
Chem 284, 18583-18587, doi:10.1074/jbc.R900003200 (2009).
30
Liu, Z., Carbrey, J. M., Agre, P. & Rosen, B. P. Arsenic trioxide
uptake by human and rat aquaglyceroporins. Biochem Biophys Res
Commun 316, 1178-1185, doi:10.1016/j.bbrc.2004.03.003 (2004).
31
Suzuki, K. T., Katagiri, A., Sakuma, Y., Ogra, Y. & Ohmichi, M.
Distributions and chemical forms of arsenic after intravenous
administration of dimethylarsinic and monomethylarsonic acids to rats.
Toxicol Appl Pharmacol 198, 336-344, doi:10.1016/j.taap.2003.10.029
(2004).
83
32
Vahter, M. Mechanisms of arsenic biotransformation. Toxicology 181182, 211-217 (2002).
33
Roy, P. & Saha, A. Metabolism and toxicity of arsenic: A human
carcinogen. Current Science 82, 38-45 (2002).
34
Thomas, D. J., Styblo, M. & Lin, S. The cellular metabolism and
systemic toxicity of arsenic. Toxicol Appl Pharmacol 176, 127-144,
doi:10.1006/taap.2001.9258 (2001).
35
Abernathy, C. United Nations Synthesis Report on Arsenic in Drinking
Water: EXPOSURE AND HEALTH EFFECTS. (2001).
<http://www.who.int/water_sanitation_health/dwq/arsenicun3.pdf>.
36
Jomova, K. et al. Arsenic: toxicity, oxidative stress and human disease.
Journal of Applied Toxicology 31, 95-107, doi:10.1002/jat.1649
(2011).
37
Gomez-Caminero, A. et al.
(United Nations Environment
Programme, International Labour Organization, World Heath
Organization, Geneva, 2001).
38
Chen, G. Q. et al. Methylated metabolites of arsenic trioxide are more
potent than arsenic trioxide as apoptotic but not differentiation
inducers in leukemia and lymphoma cells. Cancer Res 63, 1853-1859
(2003).
39
Diaz, Z. et al. A novel arsenical has antitumor activity toward As2O3resistant and MRP1/ABCC1-overexpressing cell lines.[Erratum
appears in Leukemia. 2009 Feb;23(2):431]. Leukemia 22, 1853-1863
(2008).
40
Emadi, A. & Gore, S. D. Arsenic trioxide -- An old drug rediscovered.
Blood Reviews 24, 191-199, doi:DOI: 10.1016/j.blre.2010.04.001
(2010).
41
Centeno, J. A. et al. Pathology Related to Chronic Arsenic Exposure.
Environmental Health Perspectives 110, 883-886 (2002).
84
42
Guo, H.-R., Wang, N.-S., Hu, H. & Monson, R. R. Cell Type
Specificity of Lung Cancer Associated with Arsenic Ingestion. Cancer
Epidemiology Biomarkers & Prevention 13, 638-643 (2004).
43
Bates, M. N., Smith, A. H. & Cantor, K. P. Case-Control Study of
Bladder Cancer and Arsenic in Drinking Water. American Journal of
Epidemiology 141, 523-530 (1995).
44
Sharpe, M. Deadly waters run deep: the global arsenic crisis. Journal
of Environmental Monitoring 5, 81N-85N (2003).
45
Zhu, J., Chen, Z., Lallemand-Breitenbach, V. & de The, H. How acute
promyelocytic leukaemia revived arsenic. Nat Rev Cancer 2, 705-714
(2002).
46
Haller, J. S. Therapeutic mule: the use of arsenic in the nineteenth
century materia medica. Pharm Hist 17, 87-100 (1975).
47
Antman, K. H. Introduction: The History of Arsenic Trioxide in
Cancer Therapy. The Oncologist 6, 1-2, doi:10.1634/theoncologist.6suppl_2-1 (2001).
48
Kwong, Y. L. & Todd, D. Delicious Poison: Arsenic Trioxide for the
Treatment of Leukemia. Blood 89, 3487 (1997).
49
Forkner Ce, S. T. Arsenic as a therapeutic agent in chronic
myelogenous leukemia: Preliminary report. JAMA: The Journal of the
American Medical Association 97, 3-5,
doi:10.1001/jama.1931.02730010007002 (1931).
50
Kandel Ev, L. G. CHronic arsenical poisoning during the treatment of
chronic myeloid leukemia. Archives of Internal Medicine 60, 846-866,
doi:10.1001/archinte.1937.00180050113008 (1937).
51
Sun, H., Hu, X. & Zhang, T. Ai-ling 1 treated 32 cases of acute
promyelocytic leukemia. Chin. J. Integrat. Trad. Chin. West. Med 12,
170-171 (1992).
52
Zhang, T. D. [Treatment of acute granulocytic leukemia with "Ai ling
No. 1"--clinical analysis and experimental research]. Zhong Xi Yi Jie
He Za Zhi 4, 19-20 (1984).
85
53
Soignet, S. L. et al. Complete remission after treatment of acute
promyelocytic leukemia with arsenic trioxide. N Engl J Med 339,
1341-1348, doi:10.1056/nejm199811053391901 (1998).
54
Niu, C. et al. Studies on Treatment of Acute Promyelocytic Leukemia
With Arsenic Trioxide: Remission Induction, Follow-Up, and
Molecular Monitoring in 11 Newly Diagnosed and 47 Relapsed Acute
Promyelocytic Leukemia Patients. Blood 94, 3315-3324 (1999).
55
Douer, D. ATO: the forefront of APL treatment? Blood 107, 25882589, doi:10.1182/blood-2006-01-0264 (2006).
56
Lengfelder, E., Hofmann, W. K. & Nowak, D. Impact of arsenic
trioxide in the treatment of acute promyelocytic leukemia. Leukemia
26, 433-442 (2012).
57
Ravandi, F. et al. Effective Treatment of Acute Promyelocytic
Leukemia With All-Trans-Retinoic Acid, Arsenic Trioxide, and
Gemtuzumab Ozogamicin. J Clin Oncol 27, 504-510,
doi:10.1200/jco.2008.18.6130 (2009).
58
Shen, Z. X. et al. All-trans retinoic acid/As2O3 combination yields a
high quality remission and survival in newly diagnosed acute
promyelocytic leukemia. Proc Natl Acad Sci U S A 101, 5328-5335,
doi:10.1073/pnas.0400053101 (2004).
59
Cohen, M. H. et al. Drug approval summaries: arsenic trioxide,
tamoxifen citrate, anastrazole, paclitaxel, bexarotene. Oncologist 6, 411 (2001).
60
Camacho, L. H. et al. Leukocytosis and the retinoic acid syndrome in
patients with acute promyelocytic leukemia treated with arsenic
trioxide. J Clin Oncol 18, 2620-2625 (2000).
61
Ohnishi, K. et al. Prolongation of the QT interval and ventricular
tachycardia in patients treated with arsenic trioxide for acute
promyelocytic leukemia. Ann Intern Med 133, 881-885 (2000).
86
62
Huang, S. Y. et al. Acute and chronic arsenic poisoning associated
with treatment of acute promyelocytic leukaemia. Br J Haematol 103,
1092-1095 (1998).
63
Huang, C., Ma, W. Y., Li, J. & Dong, Z. Arsenic induces apoptosis
through a c-Jun NH2-terminal kinase-dependent, p53-independent
pathway. Cancer Res 59, 3053-3058 (1999).
64
Rojewski, M. T., Baldus, C., Knauf, W., Thiel, E. & Schrezenmeier, H.
Dual effects of arsenic trioxide (As2O3) on non-acute promyelocytic
leukaemia myeloid cell lines: induction of apoptosis and inhibition of
proliferation. Br J Haematol 116, 555-563 (2002).
65
ClinicalTrials.gov. Arsenic Trioxide,
<http://www.clinicaltrials.gov/ct2/results?term=arsenic+trioxide>
(2012).
66
Amadori, S., Fenaux, P., Ludwig, H., O’Dwyer, M. & Sanz, M. Use of
arsenic trioxide in haematological malignancies: insight into the
clinical development of a novel agent. Current Medical Research and
Opinion 21, 403-411, doi:doi:10.1185/030079904X20349 (2005).
67
Röllig, C. & Illmer, T. The efficacy of arsenic trioxide for the
treatment of relapsed and refractory multiple myeloma: A systematic
review. Cancer Treatment Reviews 35, 425-430,
doi:10.1016/j.ctrv.2009.04.007 (2009).
68
Reece, D. E. Management of multiple myeloma: The changing
landscape. Blood Reviews 21, 301-314, doi:10.1016/j.blre.2007.07.001
(2007).
69
Munshi, N. C. et al. Clinical activity of arsenic trioxide for the
treatment of multiple myeloma. Leukemia 16, 1835-1837,
doi:10.1038/sj.leu.2402599 (2002).
70
Hussein, M. A. et al. Phase 2 study of arsenic trioxide in patients with
relapsed or refractory multiple myeloma. Br J Haematol 125, 470-476,
doi:10.1111/j.1365-2141.2004.04941.x (2004).
87
71
Bahlis, N. J. et al. Feasibility and correlates of arsenic trioxide
combined with ascorbic acid-mediated depletion of intracellular
glutathione for the treatment of relapsed/refractory multiple myeloma.
Clin Cancer Res 8, 3658-3668 (2002).
72
Berenson, J. R. et al. A Phase I/II Study of Arsenic
Trioxide/Bortezomib/Ascorbic Acid Combination Therapy for the
Treatment of Relapsed or Refractory Multiple Myeloma. Clin Cancer
Res 13, 1762-1768, doi:10.1158/1078-0432.ccr-06-1812 (2007).
73
Sekeres, M. A. Arsenic trioxide as a treatment for myelodysplastic
syndrome. Curr Hematol Malig Rep 1, 34-38, doi:10.1007/s11899006-0015-6 (2006).
74
List, A. F. New approaches to the treatment of myelodysplasia.
Oncologist 7 Suppl 1, 39-49 (2002).
75
Vey, N. et al. Arsenic trioxide in patients with myelodysplastic
syndromes: a phase II multicenter study. J Clin Oncol 24, 2465-2471,
doi:10.1200/jco.2005.03.9503 (2006).
76
Schiller, G. J. et al. Phase II multicenter study of arsenic trioxide in
patients with myelodysplastic syndromes. J Clin Oncol 24, 2456-2464,
doi:10.1200/jco.2005.03.7903 (2006).
77
Rea, D., Rousselot, P., Guilhot, J., Guilhot, F. & Mahon, F. X. Curing
chronic myeloid leukemia. Curr Hematol Malig Rep 7, 103-108,
doi:10.1007/s11899-012-0117-2 (2012).
78
Fabbro, D. BCR-ABL signaling: A new STATus in CML. Nat Chem
Biol 8, 228-229 (2012).
79
Kantarjian, H. et al. Hematologic and cytogenetic responses to
imatinib mesylate in chronic myelogenous leukemia. N Engl J Med
346, 645-652, doi:10.1056/NEJMoa011573 (2002).
80
Hochhaus, A. et al. Molecular and chromosomal mechanisms of
resistance to imatinib (STI571) therapy. Leukemia 16, 2190-2196,
doi:10.1038/sj.leu.2402741 (2002).
88
81
Puccetti, E. et al. BCR-ABL mediates arsenic trioxide-induced
apoptosis independently of its aberrant kinase activity. Cancer Res 60,
3409-3413 (2000).
82
Murgo, A. J. Clinical Trials of Arsenic Trioxide in Hematologic and
Solid Tumors: Overview of the National Cancer Institute Cooperative
Research and Development Studies. Oncologist 6, 22-28,
doi:10.1634/theoncologist.6-suppl_2-22 (2001).
83
Bael, T. E., Peterson, B. L. & Gollob, J. A. Phase II trial of arsenic
trioxide and ascorbic acid with temozolomide in patients with
metastatic melanoma with or without central nervous system
metastases. Melanoma Res 18, 147-151,
doi:10.1097/CMR.0b013e3282f2a7ae (2008).
84
Tarhini, A. A. et al. Safety and efficacy of arsenic trioxide for patients
with advanced metastatic melanoma. Cancer 112, 1131-1138,
doi:10.1002/cncr.23284 (2008).
85
Lin, C. C. et al. Arsenic trioxide in patients with hepatocellular
carcinoma: a phase II trial. Invest New Drugs 25, 77-84,
doi:10.1007/s10637-006-9004-9 (2007).
86
Huilgol, N. G. A phase I study to study arsenic trioxide with radiation
and hyperthermia in advanced head and neck cancer. Int J
Hyperthermia 22, 391-397, doi:10.1080/02656730600722685 (2006).
87
Gianni, M. et al. Combined arsenic and retinoic acid treatment
enhances differentiation and apoptosis in arsenic-resistant NB4 cells.
Blood 91, 4300-4310 (1998).
88
Chen, G.-Q. et al. Use of Arsenic Trioxide (As2O3 ) in the Treatment
of Acute Promyelocytic Leukemia (APL): I. As2O3 Exerts DoseDependent Dual Effects on APL Cells. Blood 89, 3345-3353 (1997).
89
Chen, G.-Q. et al. Use of Arsenic Trioxide (As2O3 ) in the Treatment
of Acute Promyelocytic Leukemia (APL): I. As2O3 Exerts DoseDependent Dual Effects on APL Cells. Blood 89, 3345-3353 (1997).
89
90
Shao, R., Davison, Miller, Wenlin, Angelika, Kelly, Wilson H. et al.
Arsenic Trioxide as an Inducer of Apoptosis and Loss of PML/RARα
Protein in Acute Promyelocytic Leukemia Cells. J Natl Cancer Inst 90,
124-133, doi:10.1093/jnci/90.2.124 (1998).
91
Bernardi, R. & Pandolfi, P. P. Structure, dynamics and functions of
promyelocytic leukaemia nuclear bodies. Nat Rev Mol Cell Biol 8,
1006-1016, doi:10.1038/nrm2277 (2007).
92
Wang, Z. G. et al. PML is essential for multiple apoptotic pathways.
Nat Genet 20, 266-272, doi:10.1038/3073 (1998).
93
Lallemand-Breitenbach, V., Zhu, J., Chen, Z. & de Thé, H. Curing
APL through PML/RARA degradation by As2O3. Trends in Molecular
Medicine 18, 36-42, doi:10.1016/j.molmed.2011.10.001 (2012).
94
Mengeling, B. J., Phan, T. Q., Goodson, M. L. & Privalsky, M. L.
Aberrant corepressor interactions implicated in PML-RAR(alpha) and
PLZF-RAR(alpha) leukemogenesis reflect an altered recruitment and
release of specific NCoR and SMRT splice variants. J Biol Chem 286,
4236-4247, doi:10.1074/jbc.M110.200964 (2011).
95
de The, H. & Chen, Z. Acute promyelocytic leukaemia: novel insights
into the mechanisms of cure. Nat Rev Cancer 10, 775-783,
doi:10.1038/nrc2943 (2010).
96
Davison, K., Cote, S., Mader, S. & Miller, W. H., Jr. Glutathione
depletion overcomes resistance to arsenic trioxide in arsenic-resistant
cell lines. Leukemia 17, 931-940 (2002).
97
Davison, K., Mann, K. K., Waxman, S. & Miller, W. H., Jr. JNK
activation is a mediator of arsenic trioxide-induced apoptosis in acute
promyelocytic leukemia cells. Blood 103, 3496-3502 (2004).
98
Dai, J., Weinberg, R. S., Waxman, S. & Jing, Y. Malignant Cells Can
Be Sensitized to Undergo Growth Inhibition and Apoptosis by Arsenic
Trioxide Through Modulation of the Glutathione Redox System. Blood
93, 268-277 (1999).
90
99
Cai, X. et al. Arsenic trioxide-induced apoptosis and differentiation are
associated respectively with mitochondrial transmembrane potential
collapse and retinoic acid signaling pathways in acute promyelocytic
leukemia. Leukemia 14, 262-270 (2000).
100
Rojewski, M. T., Korper, S., Thiel, E. & Schrezenmeier, H.
Depolarization of mitochondria and activation of caspases are common
features of arsenic(III)-induced apoptosis in myelogenic and lymphatic
cell lines. Chem Res Toxicol 17, 119-128, doi:10.1021/tx034104+
(2004).
101
Qin, D. Y., Huang, R. & Wu, T. In vitro arsenic trioxide induces
apoptosis in T cells of asthmatic patients by a Bcl-2 related
mechanism. Yao Xue Xue Bao 43, 35-43 (2008).
102
Park, W. H. et al. Arsenic trioxide-mediated growth inhibition in
MC/CAR myeloma cells via cell cycle arrest in association with
induction of cyclin-dependent kinase inhibitor, p21, and apoptosis.
Cancer Res 60, 3065-3071 (2000).
103
Larochette, N. et al. Arsenite induces apoptosis via a direct effect on
the mitochondrial permeability transition pore. Exp Cell Res 249, 413421, doi:10.1006/excr.1999.4519 (1999).
104
Hengartner, M. O. The biochemistry of apoptosis. Nature 407, 770-776
(2000).
105
Costantini, P., Jacotot, E., Decaudin, D. & Kroemer, G. Mitochondrion
as a novel target of anticancer chemotherapy. J Natl Cancer Inst 92,
1042-1053 (2000).
106
Kroemer, G. & de The, H. Arsenic trioxide, a novel mitochondriotoxic
anticancer agent? J Natl Cancer Inst 91, 743-745 (1999).
107
Zou, H., Li, Y., Liu, X. & Wang, X. An APAF-1.cytochrome c
multimeric complex is a functional apoptosome that activates
procaspase-9. J Biol Chem 274, 11549-11556 (1999).
91
108
Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The
release of cytochrome c from mitochondria: a primary site for Bcl-2
regulation of apoptosis. Science 275, 1132-1136 (1997).
109
Huang, X. J., Wiernik, P. H., Klein, R. S. & Gallagher, R. E. Arsenic
trioxide induces apoptosis of myeloid leukemia cells by activation of
caspases. Med Oncol 16, 58-64 (1999).
110
Boulares, A. H. et al. Role of poly(ADP-ribose) polymerase (PARP)
cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates
of apoptosis in transfected cells. J Biol Chem 274, 22932-22940
(1999).
111
Fulda, S. & Debatin, K. M. Extrinsic versus intrinsic apoptosis
pathways in anticancer chemotherapy. Oncogene 25, 4798-4811
(2006).
112
Kitamura, K. et al. Involvement of CD95-independent caspase 8
activation in arsenic trioxide-induced apoptosis. Leukemia 14, 17431750 (2000).
113
Pelicano, H., Carney, D. & Huang, P. ROS stress in cancer cells and
therapeutic implications. Drug Resist Updat 7, 97-110,
doi:10.1016/j.drup.2004.01.004 (2004).
114
Anderson, K. M., Seed, T., Ou, D. & Harris, J. E. Free radicals and
reactive oxygen species in programmed cell death. Medical
Hypotheses 52, 451-463, doi:10.1054/mehy.1997.0521 (1999).
115
Apel, K. & Hirt, H. Reactive oxygen species: metabolism, oxidative
stress, and signal transduction. Annu Rev Plant Biol 55, 373-399,
doi:10.1146/annurev.arplant.55.031903.141701 (2004).
116
Czarna, M. & Jarmuszkiewicz, W. [Role of mitochondria in reactive
oxygen species generation and removal; relevance to signaling and
programmed cell death]. Postepy Biochem 52, 145-156 (2006).
117
Inoue, M. et al. Mitochondrial generation of reactive oxygen species
and its role in aerobic life. Curr Med Chem 10, 2495-2505 (2003).
92
118
Davis, W., Jr., Ronai, Z. & Tew, K. D. Cellular thiols and reactive
oxygen species in drug-induced apoptosis. J Pharmacol Exp Ther 296,
1-6 (2001).
119
Chou, W. C. et al. Role of NADPH oxidase in arsenic-induced reactive
oxygen species formation and cytotoxicity in myeloid leukemia cells.
Proc Natl Acad Sci U S A 101, 4578-4583,
doi:10.1073/pnas.0306687101 (2004).
120
Sumi, D., Shinkai, Y. & Kumagai, Y. Signal transduction pathways
and transcription factors triggered by arsenic trioxide in leukemia
cells. Toxicology and Applied Pharmacology 244, 385-392, doi:DOI:
10.1016/j.taap.2010.02.012 (2010).
121
Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel
strategy to enhance drug-induced apoptosis in human leukemia cells by
a reactive oxygen species-mediated mechanism. J Biol Chem 278,
37832-37839, doi:10.1074/jbc.M301546200 (2003).
122
Nishina, H., Wada, T. & Katada, T. Physiological roles of SAPK/JNK
signaling pathway. J Biochem 136, 123-126, doi:10.1093/jb/mvh117
(2004).
123
Verma, A. et al. Activation of Rac1 and the p38 Mitogen-activated
Protein Kinase Pathway in Response to Arsenic Trioxide. J Biol Chem
277, 44988-44995, doi:10.1074/jbc.M207176200 (2002).
124
Wen, J. et al. P38 MAPK inhibition enhancing ATO-induced
cytotoxicity against multiple myeloma cells. Br J Haematol 140, 169180, doi:10.1111/j.1365-2141.2007.06895.x (2008).
125
Vives-Bauza, C., Starkov, A. & Garcia-Arumi, E. Measurements of the
antioxidant enzyme activities of superoxide dismutase, catalase, and
glutathione peroxidase. Methods Cell Biol 80, 379-393,
doi:10.1016/s0091-679x(06)80019-1 (2007).
126
Jing, Y., Dai, J., Chalmers-Redman, R. M. E., Tatton, W. G. &
Waxman, S. Arsenic Trioxide Selectively Induces Acute
93
Promyelocytic Leukemia Cell Apoptosis Via a Hydrogen PeroxideDependent Pathway. Blood 94, 2102-2111 (1999).
127
Jing, Y., Dai, J., Chalmers-Redman, R. M., Tatton, W. G. & Waxman,
S. Arsenic trioxide selectively induces acute promyelocytic leukemia
cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 94,
2102-2111 (1999).
128
Leslie, E. M., Haimeur, A. & Waalkes, M. P. Arsenic transport by the
human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a
tri-glutathione conjugate is required. J Biol Chem 279, 32700-32708,
doi:10.1074/jbc.M404912200 (2004).
129
Holmgren, A. & Bjornstedt, M. Thioredoxin and thioredoxin
reductase. Methods Enzymol 252, 199-208 (1995).
130
Arner, E. S. & Holmgren, A. Physiological functions of thioredoxin
and thioredoxin reductase. Eur J Biochem 267, 6102-6109 (2000).
131
Zhou, Y., Hileman, E. O., Plunkett, W., Keating, M. J. & Huang, P.
Free radical stress in chronic lymphocytic leukemia cells and its role in
cellular sensitivity to ROS-generating anticancer agents. Blood 101,
4098-4104, doi:10.1182/blood-2002-08-2512 (2003).
132
Biswas, S. et al. Arsenic trioxide and ascorbic acid demonstrate
promising activity against primary human CLL cells in vitro. Leuk Res
34, 925-931, doi:10.1016/j.leukres.2010.01.020 (2010).
133
Grad, J. M. et al. Ascorbic acid enhances arsenic trioxide-induced
cytotoxicity in multiple myeloma cells. Blood 98, 805-813,
doi:10.1182/blood.V98.3.805 (2001).
134
Konig, H. et al. Enhanced Bcr-Abl-specific antileukemic activity of
arsenic trioxide (Trisenox) through glutathione-depletion in imatinibresistant cells. Haematologica 92, 838-841 (2007).
135
Diaz, Z. et al. Trolox selectively enhances arsenic-mediated oxidative
stress and apoptosis in APL and other malignant cell lines. Blood 105,
1237-1245 (2005).
94
136
Mathieu, J. & Besancon, F. Arsenic trioxide represses NF-kappaB
activation and increases apoptosis in ATRA-treated APL cells. Ann N
Y Acad Sci 1090, 203-208, doi:10.1196/annals.1378.022 (2006).
137
Panwalkar, A., Verstovsek, S. & Giles, F. Nuclear factor-kappaB
modulation as a therapeutic approach in hematologic malignancies.
Cancer 100, 1578-1589, doi:10.1002/cncr.20182 (2004).
138
Gilmore, T. D. Introduction to NF-[kappa]B: players, pathways,
perspectives. Oncogene 25, 6680-6684 (2006).
139
Kapahi, P. et al. Inhibition of NF-kappa B activation by arsenite
through reaction with a critical cysteine in the activation loop of
Ikappa B kinase. J Biol Chem 275, 36062-36066,
doi:10.1074/jbc.M007204200 (2000).
140
Pearson, G. et al. Mitogen-Activated Protein (MAP) Kinase Pathways:
Regulation and Physiological Functions. Endocrine Reviews 22, 153183, doi:10.1210/er.22.2.153 (2001).
141
Tournier, C. et al. Requirement of JNK for stress-induced activation of
the cytochrome c-mediated death pathway. Science 288, 870-874
(2000).
142
Wada, T. et al. Impaired Synergistic Activation of Stress-activated
Protein Kinase SAPK/JNK in Mouse Embryonic Stem Cells Lacking
SEK1/MKK4. J Biol Chem 276, 30892-30897,
doi:10.1074/jbc.M011780200 (2001).
143
Yan, M. et al. Activation of stress-activated protein kinase by MEKK1
phosphorylation of its activator SEK1. Nature 372, 798-800 (1994).
144
Ramos, A. M. et al. Pharmacologic inhibitors of extracellular signalregulated kinase (ERKs) and c-Jun NH(2)-terminal kinase (JNK)
decrease glutathione content and sensitize human promonocytic
leukemia cells to arsenic trioxide-induced apoptosis. J Cell Physiol
209, 1006-1015, doi:10.1002/jcp.20806 (2006).
145
Chen, D., Chan, R., Waxman, S. & Jing, Y. Buthionine sulfoximine
enhancement of arsenic trioxide-induced apoptosis in leukemia and
95
lymphoma cells is mediated via activation of c-Jun NH2-terminal
kinase and up-regulation of death receptors. Cancer Res 66, 1141611423, doi:10.1158/0008-5472.can-06-0409 (2006).
146
Kajiguchi, T. et al. Sustained activation of c-jun-terminal kinase (JNK)
is closely related to arsenic trioxide-induced apoptosis in an acute
myeloid leukemia (M2)-derived cell line, NKM-1. Leukemia 17, 21892195, doi:10.1038/sj.leu.2403120 (2003).
147
Giafis, N. et al. Role of the p38 Mitogen-Activated Protein Kinase
Pathway in the Generation of Arsenic Trioxide–Dependent Cellular
Responses. Cancer Res 66, 6763-6771, doi:10.1158/0008-5472.can-053699 (2006).
148
Kang, Y. H. & Lee, S. J. The role of p38 MAPK and JNK in Arsenic
trioxide-induced mitochondrial cell death in human cervical cancer
cells. Journal of Cellular Physiology 217, 23-33,
doi:10.1002/jcp.21470 (2008).
149
Iwama, K., Nakajo, S., Aiuchi, T. & Nakaya, K. Apoptosis induced by
arsenic trioxide in leukemia U937 cells is dependent on activation of
p38, inactivation of ERK and the Ca2+-dependent production of
superoxide. Int J Cancer 92, 518-526 (2001).
150
Davis, R. J. Signal transduction by the JNK group of MAP kinases.
Cell 103, 239-252 (2000).
151
Lei, K. et al. The Bax subfamily of Bcl2-related proteins is essential
for apoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol
Cell Biol 22, 4929-4942 (2002).
152
Yoshida, K., Yamaguchi, T., Natsume, T., Kufe, D. & Miki, Y. JNK
phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl
in the apoptotic response to DNA damage. Nat Cell Biol 7, 278-285,
doi:10.1038/ncb1228 (2005).
153
Bhattacharjee, H., Carbrey, J., Rosen, B. P. & Mukhopadhyay, R. Drug
uptake and pharmacological modulation of drug sensitivity in leukemia
96
by AQP9. Biochem Biophys Res Commun 322, 836-841,
doi:10.1016/j.bbrc.2004.08.002 (2004).
154
Derijard, B. et al. JNK1: a protein kinase stimulated by UV light and
Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell 76, 1025-1037 (1994).
155
Coso, O. A. et al. The small GTP-binding proteins Rac1 and Cdc42
regulate the activity of the JNK/SAPK signaling pathway. Cell 81,
1137-1146 (1995).
156
Teramoto, H. et al. The small GTP-binding protein rho activates c-Jun
N-terminal kinases/stress-activated protein kinases in human kidney
293T cells. Evidence for a Pak-independent signaling pathway. J Biol
Chem 271, 25731-25734 (1996).
157
Porter, A. C., Fanger, G. R. & Vaillancourt, R. R. Signal transduction
pathways regulated by arsenate and arsenite. Oncogene 18, 7794-7802,
doi:10.1038/sj.onc.1203214 (1999).
158
Zha, J., Harada, H., Yang, E., Jockel, J. & Korsmeyer, S. J. Serine
phosphorylation of death agonist BAD in response to survival factor
results in binding to 14-3-3 not BCL-X(L). Cell 87, 619-628 (1996).
159
Cardone, M. H. et al. Regulation of cell death protease caspase-9 by
phosphorylation. Science 282, 1318-1321 (1998).
160
Brunet, A. et al. Akt promotes cell survival by phosphorylating and
inhibiting a Forkhead transcription factor. Cell 96, 857-868 (1999).
161
Park, H. S. et al. Akt (protein kinase B) negatively regulates SEK1 by
means of protein phosphorylation. J Biol Chem 277, 2573-2578,
doi:10.1074/jbc.M110299200 (2002).
162
Kim, A. H., Khursigara, G., Sun, X., Franke, T. F. & Chao, M. V. Akt
phosphorylates and negatively regulates apoptosis signal-regulating
kinase 1. Mol Cell Biol 21, 893-901, doi:10.1128/mcb.21.3.893901.2001 (2001).
163
Song, J. J. & Lee, Y. J. Dissociation of Akt1 from its negative
regulator JIP1 is mediated through the ASK1–MEK–JNK signal
97
transduction pathway during metabolic oxidative stress. The Journal of
Cell Biology 170, 61-72, doi:10.1083/jcb.200502070 (2005).
164
Lyons, T. R. et al. Regulation of the Pro-apoptotic scaffolding protein
POSH by Akt. J Biol Chem 282, 21987-21997,
doi:10.1074/jbc.M704321200 (2007).
165
Mann, K. K., Colombo, M. & Miller, W. H., Jr. Arsenic trioxide
decreases AKT protein in a caspase-dependent manner. Mol Cancer
Ther 7, 1680-1687 (2008).
166
Ramos, A. M. et al. Pharmacologic inhibitors of PI3K/Akt potentiate
the apoptotic action of the antileukemic drug arsenic trioxide via
glutathione depletion and increased peroxide accumulation in myeloid
leukemia cells. Blood 105, 4013-4020, doi:10.1182/blood-2004-072802 (2005).
167
Ichijo, H. et al. Induction of Apoptosis by ASK1, a Mammalian
MAPKKK That Activates SAPK/JNK and p38 Signaling Pathways.
Science 275, 90-94, doi:10.1126/science.275.5296.90 (1997).
168
Saitoh, M. et al. Mammalian thioredoxin is a direct inhibitor of
apoptosis signal-regulating kinase (ASK) 1. Embo J 17, 2596-2606,
doi:10.1093/emboj/17.9.2596 (1998).
169
Nishitoh, H. et al. ASK1 is essential for endoplasmic reticulum stressinduced neuronal cell death triggered by expanded polyglutamine
repeats. Genes Dev 16, 1345-1355, doi:10.1101/gad.992302 (2002).
170
Matsuzawa, A. et al. ROS-dependent activation of the TRAF6-ASK1p38 pathway is selectively required for TLR4-mediated innate
immunity. Nat Immunol 6, 587-592, doi:10.1038/ni1200 (2005).
171
Taniike, M. et al. Apoptosis Signal-Regulating Kinase 1/p38 Signaling
Pathway Negatively Regulates Physiological Hypertrophy. Circulation
117, 545-552, doi:10.1161/circulationaha.107.710434 (2008).
172
Takeda, K. et al. Apoptosis Signal-regulating Kinase 1 (ASK1)
Induces Neuronal Differentiation and Survival of PC12 Cells. J Biol
Chem 275, 9805-9813, doi:10.1074/jbc.275.13.9805 (2000).
98
173
Hirotani, S. et al. Involvement of Nuclear Factor-κB and Apoptosis
Signal-Regulating Kinase 1 in G-Protein–Coupled Receptor Agonist–
Induced Cardiomyocyte Hypertrophy. Circulation 105, 509-515,
doi:10.1161/hc0402.102863 (2002).
174
Tobiume, K. et al. ASK1 is required for sustained activations of
JNK/p38 MAP kinases and apoptosis. EMBO Rep 2, 222-228,
doi:10.1093/embo-reports/kve046 (2001).
175
Tobiume, K., Saitoh, M. & Ichijo, H. Activation of apoptosis signalregulating kinase 1 by the stress-induced activating phosphorylation of
pre-formed oligomer. Journal of Cellular Physiology 191, 95-104,
doi:10.1002/jcp.10080 (2002).
176
Noguchi, T. et al. Recruitment of Tumor Necrosis Factor Receptorassociated Factor Family Proteins to Apoptosis Signal-regulating
Kinase 1 Signalosome Is Essential for Oxidative Stress-induced Cell
Death. J Biol Chem 280, 37033-37040, doi:10.1074/jbc.M506771200
(2005).
177
Sayama, K. et al. Apoptosis signal-regulating kinase 1 (ASK1) is an
intracellular inducer of keratinocyte differentiation. J Biol Chem 276,
999-1004, doi:10.1074/jbc.M003425200 (2001).
178
Rubiolo, C. et al. A balance between Raf-1 and Fas expression sets the
pace of erythroid differentiation. Blood 108, 152-159,
doi:10.1182/blood-2005-09-3866 (2006).
179
Izumiya, Y. et al. Apoptosis signal-regulating kinase 1 plays a pivotal
role in angiotensin II-induced cardiac hypertrophy and remodeling.
Circ Res 93, 874-883, doi:10.1161/01.res.0000100665.67510.f5
(2003).
180
Nakamura, K. et al. Inhibitory Effects of Antioxidants on Neonatal Rat
Cardiac Myocyte Hypertrophy Induced by Tumor Necrosis Factor-α
and Angiotensin II. Circulation 98, 794-799,
doi:10.1161/01.cir.98.8.794 (1998).
99
181
Soignet, S. L. et al. United States Multicenter Study of Arsenic
Trioxide in Relapsed Acute Promyelocytic Leukemia. J Clin Oncol 19,
3852-3860 (2001).
182
Shen, Z. X. et al. Use of arsenic trioxide (As2O3) in the treatment of
acute promyelocytic leukemia (APL): II. Clinical efficacy and
pharmacokinetics in relapsed patients. Blood 89, 3354-3360 (1997).
183
Diaz, Z. et al. Trolox enhances the anti-lymphoma effects of arsenic
trioxide, while protecting against liver toxicity. Leukemia 21, 21172127 (2007).
184
Shen, S.-C. et al. Reactive oxygen species-dependent HSP90 protein
cleavage participates in arsenical As+ 3- and MMA+ 3-induced
apoptosis through inhibition of telomerase activity via JNK activation.
Toxicology and Applied Pharmacology 229, 239-251, doi:DOI:
10.1016/j.taap.2008.01.018 (2008).
185
Roussel, M. J. & Lanotte, M. Maturation sensitive and resistant
t(15;17) NB4 cell lines as tools for APL physiopathology:
nomenclature of cells and repertory of their known genetic alterations
and phenotypes. Oncogene 20, 7287-7291, doi:10.1038/sj.onc.1204863
(2001).
186
Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W.
Oncogenic ras Provokes Premature Cell Senescence Associated with
Accumulation of p53 and p16INK4a. Cell 88, 593-602 (1997).
187
Mahieux, R. et al. Arsenic trioxide induces apoptosis in human T-cell
leukemia virus type 1– and type 2–infected cells by a caspase-3–
dependent mechanism involving Bcl-2 cleavage. Blood 98, 3762-3769,
doi:10.1182/blood.V98.13.3762 (2001).
188
Bazarbachi, A. et al. Arsenic Trioxide and InterferonInduce Cell Cycle Arrest and Apoptosis in Human T-Cell
Lymphotropic Virus Type I–Transformed Cells. Blood 93, 278-283
(1999).
100
189
Rousselot, P. et al. Arsenic Trioxide and Melarsoprol Induce
Apoptosis in Plasma Cell Lines and in Plasma Cells from Myeloma
Patients. Cancer Res 59, 1041-1048 (1999).
190
Takeda, K., Noguchi, T., Naguro, I. & Ichijo, H. Apoptosis SignalRegulating Kinase 1 in Stress and Immune Response. Annual Review
of Pharmacology and Toxicology 48, 199-225,
doi:doi:10.1146/annurev.pharmtox.48.113006.094606 (2008).
191
Yan, W., Arai, A., Aoki, M., Ichijo, H. & Miura, O. ASK1 is activated
by arsenic trioxide in leukemic cells through accumulation of reactive
oxygen species and may play a negative role in induction of apoptosis.
Biochemical and Biophysical Research Communications 355, 10381044, doi:DOI: 10.1016/j.bbrc.2007.02.064 (2007).
192
Brown, J. M. & Wouters, B. G. Apoptosis, p53, and Tumor Cell
Sensitivity to Anticancer Agents. Cancer Res 59, 1391-1399 (1999).
193
Ortner, E. & Moelling, K. Heteromeric complex formation of ASK2
and ASK1 regulates stress-induced signaling. Biochemical and
Biophysical Research Communications 362, 454-459,
doi:10.1016/j.bbrc.2007.08.006 (2007).
194
Takeda, K. et al. Apoptosis Signal-regulating Kinase (ASK) 2
Functions as a Mitogen-activated Protein Kinase Kinase Kinase in a
Heteromeric Complex with ASK1. J Biol Chem 282, 7522-7531,
doi:10.1074/jbc.M607177200 (2007).
195
Morita, K. et al. Negative feedback regulation of ASK1 by protein
phosphatase 5 (PP5) in response to oxidative stress. Embo J 20, 60286036, doi:10.1093/emboj/20.21.6028 (2001).
196
Saito, J. et al. Regulation of apoptosis signal-regulating kinase 1 by
protein phosphatase 2Cepsilon. Biochem J 405, 591-596,
doi:10.1042/bj20070231 (2007).
197
Nishitoh, H. et al. ASK1 is essential for JNK/SAPK activation by
TRAF2. Mol Cell 2, 389-395 (1998).
101
198
Noguchi, T. et al. Recruitment of tumor necrosis factor receptorassociated factor family proteins to apoptosis signal-regulating kinase
1 signalosome is essential for oxidative stress-induced cell death. J
Biol Chem 280, 37033-37040, doi:10.1074/jbc.M506771200 (2005).
199
Morrison, D. K. The 14-3-3 proteins: integrators of diverse signaling
cues that impact cell fate and cancer development. Trends Cell Biol 19,
16-23, doi:10.1016/j.tcb.2008.10.003 (2009).
200
Zhang, L., Chen, J. & Fu, H. Suppression of apoptosis signalregulating kinase 1-induced cell death by 14-3-3 proteins. Proc Natl
Acad Sci U S A 96, 8511-8515 (1999).
201
Min, W. et al. AIP1 Recruits Phosphatase PP2A to ASK1 in Tumor
Necrosis Factor-Induced ASK1-JNK Activation. Circ Res 102, 840848, doi:10.1161/circresaha.107.168153 (2008).
202
Nakamura, H., Nakamura, K. & Yodoi, J. Redox regulation of cellular
activation. Annu Rev Immunol 15, 351-369,
doi:10.1146/annurev.immunol.15.1.351 (1997).
203
Holmgren, A. Thioredoxin structure and mechanism: conformational
changes on oxidation of the active-site sulfhydryls to a disulfide.
Structure 3, 239-243 (1995).
204
Mustacich, D. & Powis, G. Thioredoxin reductase. Biochem J 346 Pt
1, 1-8 (2000).
205
Lu, J., Chew, E.-H. & Holmgren, A. Targeting thioredoxin reductase is
a basis for cancer therapy by arsenic trioxide. Proceedings of the
National Academy of Sciences 104, 12288-12293,
doi:10.1073/pnas.0701549104 (2007).
206
Woodcock, J. M., Murphy, J., Stomski, F. C., Berndt, M. C. & Lopez,
A. F. The Dimeric Versus Monomeric Status of 14-3-3ζ Is Controlled
by Phosphorylation of Ser58 at the Dimer Interface. J Biol Chem 278,
36323-36327, doi:10.1074/jbc.M304689200 (2003).
207
Gromer, S., Arscott, L. D., Williams, C. H., Jr., Schirmer, R. H. &
Becker, K. Human placenta thioredoxin reductase. Isolation of the
102
selenoenzyme, steady state kinetics, and inhibition by therapeutic gold
compounds. J Biol Chem 273, 20096-20101 (1998).
208
Marzano, C. et al. Inhibition of thioredoxin reductase by auranofin
induces apoptosis in cisplatin-resistant human ovarian cancer cells.
Free Radic Biol Med 42, 872-881,
doi:10.1016/j.freeradbiomed.2006.12.021 (2007).
209
Liu, J. J. et al. Inhibition of thioredoxin reductase by auranofin induces
apoptosis in adriamycin-resistant human K562 chronic myeloid
leukemia cells. Pharmazie 66, 440-444 (2011).
210
Dong, S. et al. 14–3-3 integrates prosurvival signals mediated by the
AKT and MAPK pathways in ZNF198-FGFR1–transformed
hematopoietic cells. Blood 110, 360-369, doi:10.1182/blood-2006-12065615 (2007).
103