prevention of genomic instability and cancer by … of genomic instability and cancer by dietary...
TRANSCRIPT
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Prevention of Genomic Instability and Cancer by Dietary
Antioxidant N-acetyl Cysteine
Ramune Reliene, Ph.D.Cancer Research Center
Department of Environmental Health Sciences
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STUDY OUTLINE
•
Antioxidant: N-acetyl cysteine
•
Cancer: Lymphoma
•
Cancer Cause: ATM deficiency
•
Biological Model: Atm-/-
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•
Free radical scavenger•
Glutathione precursor
•
Prevents acetaminophen toxicity & liver failure
•
Mucolytic agent •
Effective against flu
•
Non-toxic•
Available as over-the-counter supplement
N-Acetyl Cysteine (NAC)
HS CH2
O
CH3HN
OH
O
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•
DSS-induced colon cancer in mice (Seril et al. Carcinogenesis 23, 2002)
•
NF-B and inflammatory cytokine production:
Flu-infected lung A549 cells (Geiler et al. Biochem Pharmacol 79,2010)
H.pylori-infected gastric AGS cells
(Seo et al. Ann NY Acad Sci 973, 2002)
•
IL-1-induced COX-2 expression in osteoblasts MG63
(Origuchi et al. J Lab Clin Med
136, 2000)
NAC and Inflammation
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Ataxia Telangiectasia (AT)•
“mysterious from the start-
an entity easily diagnosed on
purely clinical grounds, often by inspection-
but elusive for a long time”, Boder E, Kroc. Found. Ser 19: 1-63, 1985
• Autosomal recessive disorder
• Occurs in 1 of ~300,000 birth
•
Arises from a mutation of the ATM gene
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Clinical Phenotype of AT•
Uncoordinated or ataxic
movements•
Ocular telangiectasia
(dilated blood vessels of the eye)
• Radiosensitivity• Sterility• Endocrine abnormalities• Immunodeficiency (from EMBO Reports v. 5, 2004)
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Clinical Phenotype of AT (cont.)
There is no treatment to prevent AT
• High cancer incidence (30 -
40%): 40% of all tumors are non-Hodgkin’s lymphoma 20% are acute lymphocytic leukemia 5% are Hodgkin’s lymphoma
• The median survival 19 -
25 years
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Cellular Phenotype of AT
• Genetic instability: chromosomal breaks translocations aneuploidy telomere shortening
• Hypersensitivity to ionizing radiation
• Chronic oxidative stress
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ATM is a Protein Kinase that Responds to DNA Double-stranded Breaks
Shiloh et al. Seminars in Cancer Biology 2004 v.14
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Atm Deficient Mice Recapitulate AT
• Thymic lymphoma• Immunodeficiency• Mild ataxia • Cells display genetic
instability• Oxidative stress: catalase activity superoxide dismutase activity thioredoxin levels ROS levels
(from Nature Genetics 32, 2002)
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Oxidative Stress → Cancer
ROS
CANCERGENOMIC INSTABILITY
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Can NAC suppress genetic instability and/or cancer in Atm-/-
mice?
HYPOTHESIS
Oxidative stress is a cause of genomic instability and cancer in Atm-/-
mice
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STUDY DESIGN
•
Atm-/-
and Atm+/+ mice were chronically exposed to NAC-drinking water throughout life
40 mM NAC
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short-term study• Oxidative stress: 8-OHdG• Genomic instability: DNA deletions
long-term study • Survival • Cancer
STUDY DESIGN
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8-OHdG Determination by HPLC
min0 5 10 15 20 25
mAu
0
100
200
300
400
ADC1 A, ADC1 (07-10-07\DNA00028.D)
0.0
15 0
.060
1.4
94 1
.737
2.1
56 2
.293
2.7
23 2
.913
3.0
18 3
.554
4.1
87 5.1
76
6.6
38
8.8
92
9.9
83
14.
044
23.
547
min0 5 10 15 20 25
mAU
0
200
400
600
800
1000
1200
1400
1600
VWD1 A, Wavelength=245 nm (07-10-07\DNA00028.D)
1.6
82 2
.043
2.2
88 2
.536 3.0
36 3.3
23
4.1
02
7.6
07 8
.113
9.7
10
11.
244
20.
309
24.
581
8-OHdG
dG
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Mouse DNA Deletion Assay
Exons 1-5 6-18 6-18 19-23
70 kbpun locus
p genehomologous deletion/ pun
reversion
Exons 1-5 6-18 19-23
pun
mouse (C57BL/6J pun/pun)
Reliene et al. Methods Mol Biol 262, 2003
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retinal pigment epithelium
optical nerve head
neural retina
choroid
optical nerve
Eye-spot
70 kb DNA Deletions Result in Black Spots on the Retinal Pigment Epithelium
Dissection of the retinal pigment epithelium(eye-spot assay)
1 Eye-spot = 1 DEL event
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Oxi
dativ
e D
NA
dam
age,
8-
OH
dG/1
06 dG
NAC Suppressed Oxidative DNA Damage
0
10
20
30
40
WT AT M AT M +NAC WT +NAC
ATM+ NAC
WT+ NAC
WT ATM
p = 0.0003 p = 0.004
Reliene et al. Cancer Res 2004, v.64
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0
2
4
6
8
WT A T M AT M +NAC WT +NAC
ATM+ NAC
WT+ NAC
WT ATM
p = 0.001 p = 0.003Fr
eque
ncy
of D
NA
del
etio
ns,
No
of e
ye-s
pots
/RP
E
NAC Suppressed DNA Deletions in Atm-/-
Mice
Reliene et al. Cancer Res 2004, v.64
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10
15
20
25
30
35
0 2 4 6 8 10
ATM
ATM-NAC
Num
ber o
f 8-O
HdG
/106
dG
Number of eye-spots/RPE
The Level of Oxidative DNA Damage is Associated with the Frequency of Deletions
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single-strand DNA break
double-strand DNA break
Stalling & re-initiation of DNA replication
DNA deletion
Oxidative DNA base damage
DNA Deletions in Atm-/- Mice may Occur through Oxidative Stress
(S-phase)
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0
0.5
1
0 20 40 60 80 100
NAC Prolonged Survival of Atm-/- Mice
Survival time, weeks
Pro
porti
on s
urvi
ving
p = 0.03Log rank test
Reliene et al. DNA Repair 2006, v.5
Atm-/- NAC
Atm-/-
Atm+/+ NAC
Atm+/+
68w50w
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NAC Suppressed Lymphoma in Atm-/- Mice
0
20
40
60
80
0 20 40 60 80 100
Survival time, weeks
Inci
denc
e of
lym
phom
a, %
p = 0.02
Reliene et al. DNA Repair 2006, v.5
Atm-/- NAC
Atm-/-
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Kidney 40x
Liver 40x
Lung10x
Control mouse Mouse with lymphoma
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NAC Reduced Tumor MultiplicityLy
mph
oma
tissu
e di
strib
utio
n, %
0
20
40
60
80
100
T hymus Spleen Liver Lymph n odes Lun g Hear t K idn ey Pan cr eas St omach Duoden um Adr en al glan d
AtmAtm+NAC
Thym
us
Spl
een
Lym
ph
node
s
Lung
Live
r
Hea
rt
Kid
ney
Pan
crea
s
Sto
mac
h
Adr
enal
gl
ands
Duo
denu
m
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Inci
denc
e of
lym
phom
a, %
NAC Reduced Tumor Incidence & Multiplicity in Atm-/- Mice
0
20
40
60
80
100
1 2
Atm-/- NACAtm-/-
p = 0.02
Num
ber o
f tu
mor
s/ m
ouse
0
1
2
3
4
5
1 2
Atm-/- NACAtm-/-
p = 0.038
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SUMMARY
• Oxidative stress • Genomic instability • Longevity • Lymphoma • Metastasis
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CONCLUSION
Antioxidant therapy may be beneficial in AT patients or other disorders associated
with oxidative stress
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Our Findings were Translated to the Clinics
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ACKNOWLEDGMENTS
Robert H. SchiestlElvira FischerGregory LawsonRichard Gatti
Lymphoma Research Foundation
UCLA School of Medicine
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70 kb DNA Deletions Result in Black Spots on the Fur
Fur-spot assay
Fur-spot
Fur-spot
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Chemical Dose % mice withfur-spots
Chemical Dose % mice withfur-spots
control - 5 -
10% BaP(benzo[a]pyrene)
150 mg/kg 63%
X-rays 1 Gy 23% BEN(benzene)
100 mg/kg 27%
EMS(ethylmethane
sulfonate)
100 mg/kg 29% TCE(trichloroethylene)
200 mg/kg 32%
MMS(methylmethane
sulfonate)
100 mg/kg 25% Aroclor1221
500 mg/kg 20%
ENU(ethyl nitrosourea)
25 mg/kg 53% Aroclor1260
500 mg/kg 26%
SOA(sodium arsenate)
20 mg/kg 29% TCDD(dioxin)
2.25 g/kg 25%
Carcinogens Induce DNA Deletions in the Exposed in utero Mice
reviewed in Reliene
& Schiestl Oncogene 2003 v. 22
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Air Pollutants Induce DNA Deletions in the Exposed in utero Mice
Agent Dose Eye spots,% above control
Diesel exhaust particles
500 mg/kg/d125 mg/kg/d62 mg/kg/d
58%50%30%
Environmentaltobacco smoke 0.5 mg/m3 TPM 27%
Reliene
et al. Mutation Research 2005 v. 570