Download Oxidative Stress, DNA Repair

Cancer Care Engineering:
Studies on Oxidative Damage and SNPs
Lisa M. Kamendulis, PhD
James E. Klaunig, PhD
Indiana University
1
Question:
Is oxidative stress status altered in individuals with polyps or colorectal cancer?
What Modulates Oxidative Stress?
Dietary and lifestyle influences
high fat diet, alcohol, smoking, etc.  increased levels
Antioxidants  decreased levels
Approach
• Whole blood collected and processed
from 235 individuals for:
– Comet Analysis (Direct and Oxidative
DNA damage)
– Total Antioxidant Capacity (TEAC)
– Focused SNP analysis
Inflammation
Sources:
Mitochondria
Endothelial Cells
Immune Cells (including microglia)
Peroxisomes
Metabolism (P450) (Xenobiotics)
Antioxidants
GSH, Vitamin E, VitC,
SOD, Catalase, etc
•
Pro-oxidants
H2O2, O2-,
HO, 1O2
Samples:
– 104 controls
– 92 polyps
– 27 colon cancer
• 11 with 1 follow-up; 4 with 2 follow-up
– 12 rectal cancer
–
• 3 with 1 follow-up; 2 with 2 follow-up
SNPs evaluated only on samples from the
initial visit
2
Question:
Is oxidative stress status altered in individuals with polyps or colorectal cancer?
• Comet data (235 samples analyzed):
Group
n
Control
Polyp
Colon Cancer
Rectal Cancer
Colorectal Cancer
104
92
27
12
39
Direct DNA
Damage
1.13 ± 0.53
1.14 ± 0.54
1.16 ± 0.58
0.93 ± 0.33
1.09 ± 0.52
Oxidative
DNA Damage
1.48 ± 0.68
1.45 ± 0.57
1.77 ± 0.59*
1.82 ± 0.55*
1.79 ± 0.55*
*p < 0.05
• Total Antioxidant Capacity (TEAC) Data (235 samples analyzed):
Group
Control
Polyp
Colon Cancer
Rectal Cancer
Colorectal Cancer
n
104
92
27
12
15
Antioxidant Capacity
5.62 ± 0.47
5.71 ± 0.50
5.72 ± 0.43
5.87 ± 0.54
5.77 ± 0.46
*p < 0.05
3
Question:
Do differences exist in SNPs for oxidative stress and damage, and selective genes
associated with colorectal cancer?
SNPs evaluated (129 samples analyzed)
• Oxidative Stress
–
–
–
–
–
Catalase
SOD2
NOS3
GSTP
GSTM1
• DNA Damage/Repair
–
–
–
–
APEX1
XRCC
OGG1
TP53
• Inflammation
– IL-6
– IL-8
– PTGS2 (COX2)
• Vitamin D Status
– VDR
– CASR
– CYP24A1
• Metabolism
– CYP1A2
– CYP3A4
4
Question:
Do differences exist in SNPs for oxidative stress and damage, and selective genes
associated with colorectal cancer?
APEX1
(I64V)
II
CASR
(A986S)
Catalase
(-262 C/T)
IV VV AA AS SS
-TT
Control (57)
100 0
0
84 16
0
58 40
2
2
7
91
9
32 60 11
0
30 59 37 56
7
0
7
Polyp (52)
100 0
0
67 31
2
62 33
6
0
4
96
4
42 54 14
3
41 41 52 40
8
0
12 88
Colon Ca (15) 100 0
0
80 13
7
80 20
0
0
20 80 27 20 53 14
0
50 36 33 53 13
0
33 67
Rectal Ca (5)
0
80 20
0
40 40 20
0
0 100 0
0
33 67 20 40 40
0
0 100
--
--
--
--
-- NN NK KK
AA AG GG CC CG GG
60 40
PTGS2
(Ex10 +837)
PTGS2
-- EE ED DD SS SC CC --
--
--
0
II
IL6 (5')
--
OGG1
(S326C)
-TT
GSTP1
(I105V)
CC CT
NOS3
(E298D)
--
GSTM1
(N173K)
TT
IL8 (5')
--
CC CT
CYP24A1
(intron)
AA AG GG GG GT
100 0
--
CYP1A2 (5')
--
SOD
(A16V)
TP53 (P72R)
VV VA AA
RR RP PP
IV VV
--
--
--
AA AG GG CC CG GG
VDR
(intron)
--
--
93
XRCC
(Q399R)
--
RR RQ QQ
AA AT TT GG GT TT CC CG GG CC CT TT AA AG GG AA AG GG
CC CG GG CC CT TT
CC
CT TT
Control (57)
22 51 27 42 47 11 58 37
5
7
32 61 39 46 16 19 58 23
65 25 11 35 42 23
51
44
5
Polyp (52)
22 51 27 37 54 10 48 48
4
4
44 52 40 46 13 25 46 29
46 44 10 31 63
6
44
50
6
Colon Ca (15) 20 40 40 53 40 7 87 7
7
20 33 47 40 40 20 20 53 27
53 33 13 40 40 20
27
47 27
Rectal Ca (5) 60
20 20 60 20 80
20 60 20 20 60 20
60
40
0
40 60 40
0
80
0
0
20
0
60 40
0
5
Question:
Do differences exist in SNPs for oxidative stress and damage, and selective genes
associated with colorectal cancer?
Control
Polyp
Colon Cancer
Rectal Cancer
Control
Polyp
Colon Cancer
Rectal Cancer
Control
Polyp
Colorectal Cancer
Rectal Cancer
n
57
52
15
5
GSTM1 (N173K)
(%)
Al-1 AL-2 Both Null
11
30
0
59
14
41
3
41
14
50
0
36
0
33
0
67
n
57
52
15
5
COX2 (-1195)
(%)
Al-1 AL-2 Both
7
61
32
4
52
44
20
47
33
20
20
60
n
57
52
15
5
APEX1 (I64V)
(%)
Al-1 AL-2 Both
100
0
0
100
0
0
100
0
0
100
0
0
•
GSTM1 null shown to be protective, %
null population decreases in colorectal
cancer
•
Polymorphism in promoter region of
COX2 reported to change
transcriptional activity. Not previously
reported in colon cancer
•
I64V amino acid change, function
unknown. No differences observed in
this population.
6
Next Steps
• Complete sample and data analysis
– 3 additional samples collected
– SNPs completed on all 235 samples
• Data Analysis/Evaluation
– SNP tools
– Correlation analysis with other endpoints
7