Dr. Mikkelsen Chronic Inflammation and Cancer

9/23/2014
Chronic Inflammation and Cancer
Reactive Oxygen/Nitrogen Species are
Hallmarks of Chronic Inflammation
• ROS/RNS produced as a normal product of
cellular metabolism include O2-, H2O2,
HOCl, OH•, ONOO• Traditional view—oxidative stress, cell
damage, aging;
• But also at low controllable levels can act
as regulators of physiological processes
Nitric Oxide Synthase Contributes to
ROS/RNS Generation
NOS produces NO in a normal (non inflammatory) environment. NO plays a
role in vasoregulation, wound healing and immune response.
Nitric Oxide Synthase Contributes to
ROS/RNS Generation
• However, in inflammatory conditions, BH4 is oxidized, leading to NOS
“uncoupling,” and generation of O2- and ONOO-.
• These ROS/RNS further create an oxidative environment, BH4
BH2more uncoupling…
• Endothelial dysfunction and poor wound repair as found in diabetic
patients
• Nitration of IκB and activation
of pro-inflammatory NFκB
• Activation of PP2A, downregulation of BRCA1 expression
and enhanced chromosomal
instability
• Nitration of Akt activation of this
cytoprotective pathway
• Tumor cells have low PKG activity; e.g. BrCa cells
express high levels of 5’PDE
• Over-expression of PKG, or treatment with sidenafil or
cell permeable cGMP are cytotoxic to BrCa and CRC.
BH4:BH2
BH4:BH2
+ SP
Normal mouse tissue
Colon
7.1±0.6
n/d
Lung
11.1±2.4
n/d
Liver
8.3±0.8
n/d
Kidney
13.4±1.2
n/d
In vitro tumor cell lines
A431
0.3±0.5
2.1±0.6
MCF7
0.6±0.2
10.1±0.4
MDA231
1.0±0.1
5.8±0.2
HT29
1.1±0.4
2.8±0.1
MCF10A
1.2±0.2
3.1±0.3
MCF7-GCH1
3.6±0.2
n/d
In vivo tumor xenografts
Fadu
0.8±0.2
2.6±0.3
MCF7
1.1±0.1
7.9±0.2
MDA231
1.4±0.3
6.4±0.4
Paired MMTVneu mouse tissue
Mammary
3.8±0.5
n/d
Fat Pad
(n=4)
Mammary
1.0±0.2
2.8±0.5
Tumors
(n=8)
(n=8)
Mouse Colon Epithelium
AOM/DSS
1.4±0.3
4.6±0.9
CRC
(n=6)
(n=6)
Paired human colon tissue
Human
4.5
n/d
Colon
(3.5-6.1)
Human
2.3
n/d
CRC
(1.5-3.5)
BH4 and BH2 have equal affinity for NOS and
thus BH4:BH2 will determine whether NOS
produces NO or RNS/ROS
NOS Uncoupling and Tumor Progression
NOS activity
ONOO- synthase (uncoupled)
NOS activity (coupled)
NO•
Cys S-nitrosylation
(or Cys oxidation by RNS):
PTPs
Caspases
Zn Proteins
NF-κB
Bcl-xl
NO• + O2- = RNS
Heme Binding Proteins:
Cytochrome C Oxidase
Soluble Guanylate Cyclase—PKG
VASP-endothelial migration and vasculogenesis
p21-cell cycle regulation
β-catenin down regulation
Tyrosine Nitration:
IB
Bcl-xl
p53 (
Keap1/Nrf2
PKCε
PP2A
Akt
1
9/23/2014
SP Recouples NOS in BrCa
SP TX inhibits Inflammatory and Anti-apoptotic
Pathways in Breast and Colon Cancer Cells
• SP in tissue culture or by inclusion in the drinking water
of mice increases NOS –inhibitor sensitive cGMP levels
and endogenouse PKG activity
• SP treatment blocks NOS inhibitor superoxide generation
• SP treatment blocks endogenous protein Tyr nitration
• Decreases β-catenin expression and down stream signaling
• Inhibits basal NF-κB transcriptional activity
• Inhibits recruitment of inflammatory cells to tumors
• Inhibits expression of pro-inflammatory cytokines
• In some tumors enhances expression of CDK inhibitors p21
and p27
SP Inhibits Proliferation of MMTVneu Tumor Cells
Inducible Colorectal Cancer Model
B.
Mouse
ID
Tumor
number
M001
M001
M002
M003
M004
M004
M005
M005
M006
T1
T2
T3
T4
T5
T6
T7
T8
T9
Tumor Samples
Body
Wt. (g)
Pre SP
Body
Wt. (g)
Post SP
26.1
25.8
27.7
26.1
28.8
28.7
26.3
25.8
26.5
28.7
26.2
26.8
Pre-SP %ID/g
± STD
Total Average (n=9)
7.28 ± 2.50
Censored Average (n=7) 7.51 ± 2.48
%ID/g
Post SP
11.440
10.990
7.453
5.785
5.178
6.353
4.701
5.499
7.141
9.305
7.447
4.671
5.175
4.941
1.782
4.683
7.343
6.457
Post-SP %ID/g
± STD
5.76 ± 2.17
5.66 ± 2.38
%
Change
in FDG
Uptake
-18.7
-32.2
-37.3
-10.6
-4.6
-72.0
-0.4
+33.5
-9.6
Figure 1
Matched
2-tail t-test
p = 0.062
p = 0.019
Days
D.
A.
Disease Ac vity Index
C.
%ID/g
Pre SP
B.
1 mm
1 mm
C.
Rela ve Body Weight
A.
D..
D.
Days
SP Decreased Inflammation in AOM/DSS Induced Colitis
Prophylactic SP decreases CRC tumorigenesis
Rela ve Expression
&
IL-6
• Reduced protein Tyr nitration
Relative Tumor Volume
• Also decreased infiltration of tumors
with macrophages and neutrophils
Figure 5
Tumors/Unit Length
IL-1b
A.
B.
&
AOM +DSS
&&
AOM+DSS+SP
AOM +DSS
AOM+DSS+SP
N=10 for each group * p<.05
IL-17a
&& & &
C DSS DSS SP
+SP
2
9/23/2014
SP Decreases FDG Uptake
SP Decreased β-Catenin Expression in Tumors
Acknowledgements
Mikkelsen Lab:
Christopher Rabender, PhD, post doctoral fellow
Robert Cardnell, PhD, postdoctoral fellow
Asim Alam, BS (MD,PhD Student)
Vasily Yakovlev, MD,PhD
Center for Molecular Imaging
Jamal Zweit
Sundaresan Gobalakrishnan
Li Wang
3

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