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Larry W. Oberley_ Ph.D - Univers

VIEWS: 22 PAGES: 64

									The Mechanism of the Tumor Suppressive Effects of MnSOD Overexpression
Larry W. Oberley, Ph.D.
Free Radical and Radiation Biology Program Department of Radiation Oncology University of Iowa

Current Collaborators
• Shawn Flanagan – Assistant Research Scientist • Wenqing Sun – Research Assistant III • Yuping Zhang – Research Assistant II • Matthew Zimmerman – Postdoctoral Scholar • Ehab Sarsour – Predoctoral Student • Jingriu Liu – Predoctoral Student • Ling Xiao – Predoctoral Student • Changbin Du –Predoctoral Student • Suwimol Jetawattana - Predoctoral Student

Ph.D. Students
• • • • • • • Isabel B. Bize Susan W.C. Leuthauser Shailendra K. Sahu Daret Kasemset St.Clair Douglas R. Spitz, Jr. Elaine Sierra-Rivera Michael L. McCormick

Ph.D. Students (continued)
• • • • • • • Yi Sun S. Thomas Deahl, III James H. Elwell Gregg A. Cohen Jian Jian Li Lisa A. Ridnour Weixiong Zhong

Ph.D. Students (continued)
• • • • • • • Rugao Liu Jarunee Thongphasuk Ernest Wing Ngai Lam Jianglan Hannah Zhang Shijun Li Ji-Qin Yang Ying Zhang

Ph.D. Students (continued)
• • • • • Meredith Preuss Nick Khoo Christine Weydert Wenqing Sun Min Wang

Postdoctoral Fellows
• • • • • • Garry R. Buettner Susan W.C. Leuthauser Dean P. Loven James H. Elwell Kirk Baumgardner Tao Yan

Postdoctoral Fellows (continued)
• • • • • • Weixiong Zhong Rugao Liu Shawn Flanagan Dan DeArmond Matthew Zimmerman Christine Weydert

Long-time Collaborator
Terry D. Oberley, M.D., Ph.D. Department of Pathology The University of Wisconsin Madison, Wisconsin

Antioxidant Enzymes
The antioxidant enzymes are proteins with antioxidant properties. There are three known classes of antioxidant enzymes: • Superoxide dismutases • Catalases • Peroxidases
There are many forms of each class of protein. In general, cancer cells have low levels of these enzymes, when compared to an appropriate normal cell control.

Primary Antioxidant Enzyme System

SOD in Eukaryotic Cells
MW / Da MnSOD CuZnSOD
ECSOD ECMnSOD

Location Mitochondria Cytosol, nucleus
Extracellular Extracellular

88,000 32,000
135,000 150,000

MnSOD: A new type of tumor suppressor gene

Generality of Loss of MnSOD
Diminished amounts of MnSOD have been observed in:
• • • • • • • • Spontaneous tumors Transplanted tumors Virally induced tumors Chemically induced tumors Hormonally induced tumors In vitro and in vivo tumors All species examined 90% of cancer types have low MnSOD, while 10% have high MnSOD

Evidence for MnSOD as a Tumor Suppressor Gene
1. Diminished MnSOD protein in cancer due to: • Loss of heterozygosity for MnSOD • Abnormal methylation of MnSOD promoter • Mutations in MnSOD promoter • Mutations in structural gene 2. Overexpression of MnSOD protein results in inhibition of cancer cell growth both in vitro and in vivo

Paradoxical effects of thiol reagents on Jurkat cells and a new thiol-sensitive mutant form of human mitochondrial superoxide dismutase

Daniel Hernandez-Saavedra and Joe M. McCord
Webb-Warring Institute for Cancer, Aging, and Antioxidant Research, University of Colorado Health Sciences Center, Denver, Colorado 80262

Cancer Research 63:159-163

January 1, 2003

Effect of MPG on the Specific Activity of SOD in the Jurkat T-cell line

cDNA Sequence From Wild-type HPBL; Both Jurkat T-Cell Line sod2 Gene Alleles

Comparison of effects of two polymorphic variants of manganese containing superoxide dismutase on human breast MCF-7 cancer cell phenotype
Cancer Res. 59(24):6276-6283, 1999.

Nomenclature
WT
SOD

Parental MCF-7 cells
Clones transfected with Thr58 MnSOD cDNA. The clones were named SOD15, SOD18, SOD23, and SOD50.

Neo4 A clone transfected with vector plasmids

Mn

Clones transfected with Ile58 MnSOD cDNA. The clones were named Mn1, Mn11, Mn28, Mn40, Mn44, Mn52, Mn59, and Mn63.

MnSOD Western Blot

WT Neo SOD SOD SOD SOD Mn Mn 4 15 18 23 50 1 11

Mn 28

Mn 40

Mn Mn 44 52

Mn Mn 59 63

Integrated density value (arbitrary units)
10000 12000 14000 2000 * * * * * * * * * * * * * * * * * * * * * * 4000 6000 8000 0 *

W T Ne o SO 4 D1 SO 5 D1 SO 8 D2 SO 3 D5 0 M n1 M n1 1 M n2 8 M n4 0 M n4 4 M n5 2 M n5 9 M n6 3

MnSOD activity (U/mg protein)

100

150

200

250

300

50
7 7 * 19 * 11 10 * 23 * 62 * 142 * 57 * 44 * * 44 53

0
* 52

* 230

W T N e S O o4 D S O 15 D S O 18 D S O 23 D 50 M n1 M n1 1 M n2 8 M n4 0 M n4 4 M n5 2 M n5 9 M n6 3

WT Neo
Relative IDV (fold) Relative Activity (fold)

SOD SOD SOD SOD Mn Mn Mn Mn Mn Mn Mn Mn 15 18 23 50 1 11 28 40 44 52 59 63

1 1

1 1

9 3

5 2

4 1

9 3

9 9

20 19

7 8

33 31

7 6

5 6

8 7

8 7

40
Relative MnSOD Activity (fold)

30

SOD Mn

20 y = 0.925x + 0.412 r = 0.970 10 y = 0.345x - 0.111 r = 0.942 0 0 10 20 30 40 Relative IDV (fold)

250

200

Tumor Volume (mm3)

150

100

WT Neo4 SOD15 SOD18 SOD23 SOD50 Mn1 Mn28 Mn40 Mn59

WT Neo4

4/4 3/4

SOD15 SOD18
SOD23 SOD50

1/4 3/4
2/4 1/4

Mn1 Mn11
Mn28 Mn40

1/4 0/4
1/4 2/4

50

Mn44 Mn52
Mn59

0/4 0/4
1/4 0/4

0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mn63

Time (weeks)

Conclusions
• Clones with high MnSOD protein, but low enzymatic activity, had much less tumor suppressive effect than clones with comparable levels of MnSOD protein, but high enzymatic activity • Therefore, active MnSOD is necessary for a strong tumor suppressive effect

Human Oral Squamous Cell Carcinoma as a Typical Example
• The human oral squamous carcinoma cell line SCC-25 was transfected with MnSOD cDNA. Several clones overexpressing MnSOD were isolated and characterized. Human Gene Therapy 8(5):585595, 1997.

Vector Control, and MnSOD Transfected Cells
Cell Lines
P

Antioxidant Enzyme Activity in Parental,
MnSOD
(U/mg protein)

Total SOD
(U/mg protein)

CuZnSOD
(U/mg protein)

Catalase
(k/mg protein)

GPx
(mU/mg protein)

47  6

31  5

16  8

106  10

11  2

V1
V2

43  5
54  4

28  2
33  4

15  5
21 5

115  12
102  10

91
10  1

Mn1
Mn2

80  5
112  9

64  2*
95  6*

16  5
17  10

119  5
112  5

18  2*
21  3*

Mn3 Mn4 Mn5 Mn6

74  5 136  6 85  5 184  9

62  6* 116  4* 71  8* 158  7*

13  8 20  7 14  8 26  12

98  8 106  6 102  7 114  10

18  1* 23  2* 19  2* 21  2*

Note: The results were expressed as mean  SEM of 3 individual measurements

* Indicating significantly different from parental cell line p < 0.05

Conclusions
• In most cancer cell types, overexpression of MnSOD leads to inhibition of cell growth • In general, the higher the MnSOD activity, the greater the tumor suppressive effect • Other proteins are induced after overexpression of MnSOD

Mechanism
1. In most cancer cells, overexpression of MnSOD causes no cell killing via necrosis, apoptosis, or inflammation. 2. Growth inhibition appears to be due to cell cycle perturbation. 3. Are changes due to the reduction in the levels of superoxide radicals or an increase in the levels of hydrogen peroxide? Is NO• involved?

peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase
Shijun Li, Tao Yan, Ji-Qin Yang, Terry D. Oberley and Larry W. Oberley
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa College of Medicine, Iowa City, Iowa 52242

The role of cellular glutathione

Cancer Research 60:3927-3939

July 15, 2000

Cell Lines
Parental SOD2 Neo Zeo35 S-GPXs
U118 

Human glioma U118-9 cells A MnSOD over-expressing transfectant A vector control for SOD2 A vector control for MnSOD-GPX double transfectants MnSOD-GPX double transfectants
U118-9 + Neo  SOD2 + Zeo35  SGPXs

Native Immunoblotting for GPX1

Measured GPX Activity (mU/mg protein)

Cell Line

Tumor Incidence

Cell line
P
Incidence % 57.1

Neo
62.5

SOD 2 25

Zeo3 5 37.5

S– GPX70 25

S– GPX86 75

S– GPX146 85.7

4/7

5/8

2/8

3/8

2/8

6/8

6/7

Tumor incidence % =
(number of mice with tumor/total number of total mice) x 100.

Conclusion

The major effector of the tumor suppression effect of MnSOD is H2O2 in this cancer cell line.

Regulation of Gene Expression by MnSOD Overexpression

manganese-containing superoxide dismutase in human breast cancer cells

Inhibition of AP-1 and NF-kB by

Jian-Jian Li, Larry W. Oberley, Ming Fan, and Nancy H. Colburn
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa College of Medicine, Iowa City, Iowa 52242

The FASEB Journal 12:1713-1723

December 1998

cells overexpressing manganese-containing superoxide dismutase
Zhongkui Li, Alexander Khaletskiy, Jianyi Wang, Jeffrey Y.C. Wong, Larry W. Oberley and Jian-Jian Li
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa College of Medicine, Iowa City, Iowa 52242

Genes regulated in human breast cancer

Free Radical Biology & Medicine 30(3):260-267

2001

Up-regulated Genes in MCF-7 Cells Overexpressing MnSOD
GenBank Accession # J02958 S40706 M38690

Gene name
MET

Function oncogene

SOD/MC F (Fold*) 42

Growth arrest and DNA-damageinducible protein (GADD153)
CD9 A-catenin (cadherin-associated protein) Plakoglobin (desmoplakin III)

DNA damage  33 response
cell adhesion  7 cell-cell  2 interaction cell-cell 14 interaction

D13866
M23410

*Results represent the average of two separate microarray hybridizations and values less then 2-fold are not shown

Down-regulated Genes in MCF-7 Cells Overexpressing MnSOD
GenBank Accession # Gene name L33264 cdc2-related kinase X59798 cyclin dl (bcl-1 oncogene)

Function cell cycle regulator cell cycle regulator intermediate filament M34225 cytokeratin 8 (ck 8) markers Y09392 WSL-LR (Apo-3) apoptosis TNF-a converting enzyme U69611 apoptosis L07541 Activator-1 38 kd subunit (rfc38) DNA damage response M35410 IGFBP2 receptor AF000974 zyxin related protein (zrp-1) cell adhesion vascular endothelial growth U01134 angiogenesis regulator factor receptor 1 precursor U36223 fibroblast growth factor-8 (fgf-8) growth factor IL-1b K02770 cytokines
2-fold are not shown. ** No signal was detected in the MnSOD transfectants.

MCF/SOD (Fold*) 2 2 6 3 24 8 2 7

** 31 33

*Results represent the average of two separate microarray hybridizations and values less then

Which genes or proteins regulated by MnSOD are important?: HIF-1 may be crucial because it controls tumor proliferation and angiogenesis

Hypoxic (1% O2) Accumulation of HIF-1 Protein is Suppressed by Plasmid Transfection of MnSOD

HIF-1
Neo WT
1 1

SOD23

SOD18

SOD15

SOD50

Mn44

Mn52

Mn59

Mn63

Mn28

Mn1

Mn11

Clones

MnSOD Activity
-tubulin

1

2

3

3

6

6

7

7

8

9

1 9

Increased MnSOD Activity by Adenovirus Infection

MOI
MnSOD

0 LacZ 1.25 2.5

5

20 50 75 100 200

CuZnSOD

Hypoxic (1% O2) Accumulation of HIF-1 Protein is Suppressed by Adenoviral Transduction of MnSOD

0 MOI 21% O2 HIF-1
AdMnSOD MOI

1% O2

Regression Lines Showing a Biphasic Effect
6

HIF-1/tubulin

3

R = 0.7771 P= 0.02

2

R2 = 0.9752 P< 0.001

0 0

Relative MnSOD activity (-fold)

5

10

15

20

Vascular Endothelial Growth Factor (VEGF)

•

VEGF is an essential factor mediating new blood vessel formation and angiogenesis. Injection of VEGF into rat skin induces angiogenesis. VEGF gene expression is regulated by HIF-1 during hypoxia.

•

•

VEGF Production Increased When Cells Were Exposed to Hypoxia
3500

VEGF (pg/106 cells)

3000 2500 2000 1500 1000 500 0 0

hyp. medium no-hyp. medium hypoxia lysate

4

8

12

16

20

24

28

time in hypoxia (h)

MnSOD Suppressed Hypoxic Induction of VEGF in MCF-7 Cells
4000 3500

VEGF (pg/106 cells)

3000 2500 2000 1500 1000 500 0 0

WT-hypoxia SOD50-hypoxia Mn11-hypoxia

4

8

12

16

20

24

28

32

time in hypoxia (h)

Conclusions
• Medium levels of MnSOD overexpression led to dramatic inhibition of levels of HIF-1 protein, while large overexpression of MnSOD allowed HIF1 to accumulate. • MnSOD overexpression led to reduction in secreted VEGF protein.

Conclusions
1. MnSOD levels are low in cancer cells due to a variety of reasons. 2. Overexpression of MnSOD inhibits cancer cell growth both in vitro and in vivo. 3. The growth inhibitory effects of MnSOD overexpression in human glioma cells are mainly due to hydrogen peroxide. 4. MnSOD overexpression inhibits cell growth due to its effects on signal transduction.

Conclusions (continued)
5. A major effect of MnSOD overexpression is the lowering of HIF-1 protein levels. 6. Besides affecting cancer cell proliferation, MnSOD overexpression probably also affects angiogenesis.

ANTITUMOR THERAPIES BASED ON ANTIOXIDANT MODULATION
A vision for the future

Reactive Oxygen Species and Antioxidant Schematic Diagram
L-Glutamate -GCS BSO

-Glutamyl-cysteine
GS H2 O GPX H2O 2 SOD O2
-

BCNU NADPH GSH GR GSSG

6-Phosphogluconolactone G-6-PD Glucose6-phosphate

CAT O2 + H2O AT

NADPDHEA

Deoxyglucose Glucose

Mechanism of H2O2 Increase with BCNU and AT Addition
O
2

H2O2
GPx

H2O2

BCNU catalase

H2O2

AT

Inhibition of Oral Cancer Cell Growth by AdenovirusMnSOD plus BCNU Treatment

Christine J. Darby Weydert*, Benjamin B. Smith*, Linjing Xu†, Kevin C. Kregel†, Justine M. Ritchie‡, Charles S. Davis‡, and Larry W. Oberley*.
*Free Radical and Radiation Biology Program, Department of Radiation Oncology, Roy J. and Lucille A. Carver College of Medicine and Holden Comprehensive Cancer Center; †Department of Exercise Science, College of Liberal Arts and Sciences; and ‡Department of Biostatistics, College of Public Health and Holden Comprehensive Cancer Center, The University of Iowa, Iowa City 52242

AdMnSOD Plus BCNU Decreased Oral Cancer Growth In Vivo
A
Tumor Volume (mm3)
120

SCC-25

C
400 300 200 100 0

HCPC-1

Tumor Volume (mm3)

80

40

0
13 19 25 31 37 43 49 55 61 67 73 1 7

8 12

16 20

24 28

32 36

40 44
226 251

B
Percent Survival (%)

Time (days)

Percent Survival (%)

120

D120
80

Time (days)
**p<0.001 vs. control or BCNU

80
* ** *p<0.03 vs. control

48
** **

40
**p<0.005 vs. BCNU

40

26

51

76

101

126

151

176

201

276

130

173

216

259

302

346

389

432

475

518

561

604

44

87

Days Till Sacrifice
Control BCNU (30 mg/kg) AdMnSOD (10E9 pfu) AdMnSOD (10E9 pfu)+BCNU (15 mg/kg)

Days Till Sacrifice
Control BCNU (30 mg/kg) AdMnSOD (10E9 pfu) AdMnSOD (10E9 pfu)+BCNU (15 mg/kg)

301

1

1

0

0

4

Conclusion
• It should be possible to use MnSOD overexpression in human cancer therapy!

Thank you!


								
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