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Cancer Care Engineering:Studies on Oxidative Damage and SNPs

Lisa M. Kamendulis, PhDJames E. Klaunig, PhD

Indiana University

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Question:Is oxidative stress status altered in individuals with polyps or colorectal cancer?

Approach• Whole blood collected and processed

from 235 individuals for:– Comet Analysis (Direct and Oxidative

DNA damage)– Total Antioxidant Capacity (TEAC)– Focused SNP analysis

• 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

What Modulates Oxidative Stress?

Dietary and lifestyle influenceshigh fat diet, alcohol, smoking, etc. increased levelsAntioxidants decreased levels

Inflammation

Sources: Mitochondria Endothelial Cells Immune Cells (including microglia) Peroxisomes Metabolism (P450) (Xenobiotics)

AntioxidantsGSH, Vitamin E, VitC,

SOD, Catalase, etc

Pro-oxidants H2O2, O2

-, HO, 1O2

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• Comet data (235 samples analyzed):

• Total Antioxidant Capacity (TEAC) Data (235 samples analyzed):

Group n Direct DNA Damage

Oxidative DNA Damage

Control 104 1.13 ± 0.53 1.48 ± 0.68 Polyp 92 1.14 ± 0.54 1.45 ± 0.57 Colon Cancer 27 1.16 ± 0.58 1.77 ± 0.59* Rectal Cancer 12 0.93 ± 0.33 1.82 ± 0.55* Colorectal Cancer 39 1.09 ± 0.52 1.79 ± 0.55* *p < 0.05

Group n Antioxidant Capacity Control 104 5.62 ± 0.47 Polyp 92 5.71 ± 0.50 Colon Cancer 27 5.72 ± 0.43 Rectal Cancer 12 5.87 ± 0.54 Colorectal Cancer 15 5.77 ± 0.46 *p < 0.05

Question:Is oxidative stress status altered in individuals with polyps or colorectal cancer?

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Question:Do differences exist in SNPs for oxidative stress and damage, and selective genes associated with colorectal cancer?

• 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

SNPs evaluated (129 samples analyzed)

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APEX1 (I64V)

CASR (A986S)

Catalase (-262 C/T)

CYP1A2 (5')CYP24A1

(intron)GSTM1 (N173K)

 GSTP1 (I105V)

IL6 (5')

II IV VV AA AS SS -- -- -- -- -- -- -- -- -- NN NK KK   II IV VV -- -- --

AA AG GG GG GT TT CC CT TT CC CT TT AA AG GG CC CG GG -- AA AG GG CC CG GG

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 93

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) 100 0 0 80 20 0 40 40 20 0 0 100 0 60 40 0 0 33 67 20 40 40 0 0 100

   

IL8 (5')NOS3

(E298D)OGG1

(S326C)PTGS2

PTGS2 (Ex10 +837)

SOD (A16V)

 TP53

(P72R)VDR (intron)

XRCC (Q399R)

-- -- -- EE ED DD SS SC CC -- -- --       VV VA AA   RR RP PP -- -- -- 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 0 40 60 40 0 80 0 20 20 60 20 80 0 20 0 60 40   20 60 20 20 60 20 60 40 0

Question:Do differences exist in SNPs for oxidative stress and damage, and selective genes associated with colorectal cancer?

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Question:Do differences exist in SNPs for oxidative stress and damage, and selective genes associated with colorectal cancer?

• 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.

GSTM1 (N173K) (%)

n Al-1 AL-2 Both Null Control 57 11 30 0 59 Polyp 52 14 41 3 41 Colon Cancer 15 14 50 0 36 Rectal Cancer 5 0 33 0 67

COX2 (-1195)

(%) n Al-1 AL-2 Both

Control 57 7 61 32 Polyp 52 4 52 44 Colon Cancer 15 20 47 33 Rectal Cancer 5 20 20 60

APEX1 (I64V)

(%) n Al-1 AL-2 Both

Control 57 100 0 0 Polyp 52 100 0 0 Colorectal Cancer 15 100 0 0 Rectal Cancer 5 100 0 0

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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