The Mechanism of the Tumor Suppressive Effects of MnSOD Overexpression

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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
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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

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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

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Ph.D. Students (continued)

• Yi Sun  
• S. Thomas Deahl, III  
• James H. Elwell
• Gregg A. Cohen
• Jian Jian Li
• Lisa A. Ridnour
• Weixiong Zhong

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Ph.D. Students (continued)

• Rugao Liu
• Jarunee Thongphasuk
• Ernest Wing Ngai Lam
• Jianglan Hannah Zhang
• Shijun Li
• Ji-Qin Yang
• Ying Zhang

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Ph.D. Students (continued)

• Meredith Preuss
• Nick Khoo
• Christine Weydert
• Wenqing Sun
• Min Wang

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Postdoctoral Fellows

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

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Postdoctoral Fellows (continued)

• Weixiong Zhong
• Rugao Liu
• Shawn Flanagan
• Dan DeArmond
• Matthew Zimmerman
• Christine Weydert

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Long-time Collaborator

• Terry D. Oberley, M.D., Ph.D.
• Department of Pathology
• The University of Wisconsin
• Madison, Wisconsin

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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.
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Primary Antioxidant Enzyme System
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SOD in Eukaryotic Cells
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MnSODA new type of tumor suppressor gene
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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
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Evidence for MnSOD as a Tumor Suppressor Gene

• 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

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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 63159-163 January 1, 2003
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Effect of MPG on the Specific Activity of SOD in
the Jurkat T-cell line
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cDNA Sequence From Wild-type HPBL Both Jurkat
T-Cell Line sod2 Gene Alleles
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Comparison of effects of two polymorphic variants
of manganese containing superoxide dismutase on
human breast MCF-7 cancer cell phenotypeCancer
Res. 59(24)6276-6283, 1999.
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Nomenclature

• WT Parental MCF-7 cells
• Neo4 A clone transfected with vector plasmids
• SOD Clones transfected with Thr58 MnSOD cDNA. The
  clones were named SOD15, SOD18, SOD23, and SOD50.
• Mn Clones transfected with Ile58 MnSOD cDNA. The
  clones were named Mn1, Mn11, Mn28, Mn40, Mn44,
  Mn52, Mn59, and Mn63.

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MnSOD Western Blot
WT Neo SOD SOD SOD SOD Mn Mn Mn Mn Mn Mn Mn Mn
4 15 18 23 50 1 11 28 40 44 52 59 63
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Integrated density value (arbitrary units)
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MnSOD activity (U/mg protein)
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Tumor Volume (mm3)
Time (weeks)
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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

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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 over-expressing MnSOD were isolated and
  characterized.
•      Human Gene Therapy 8(5)585-595, 1997.

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Antioxidant Enzyme Activity in Parental, Vector
Control, and MnSOD Transfected Cells
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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

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Mechanism

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

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The role of cellular glutathione 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

Cancer Research 603927-3939 July 15, 2000
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Cell Lines

• Parental Human glioma U118-9 cells
• SOD2 A MnSOD over-expressing transfectant
• Neo A vector control for SOD2
• Zeo35 A vector control for MnSOD-GPX double
  transfectants
• S-GPXs MnSOD-GPX double transfectants

U118 ? U118-9 ? SOD2 ? SGPXs Neo
Zeo35
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Native Immunoblotting for GPX1
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Measured GPX Activity(mU/mg protein)
Cell Line
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Tumor Incidence
Tumor incidence (number of mice with
tumor/total number of total mice) x 100.
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Conclusion

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

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Regulation of Gene Expression by MnSOD
Overexpression
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Inhibition of AP-1 and NF-kB by
manganese-containing superoxide dismutase in
human breast cancer cells
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 121713-1723 December 1998
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Genes regulated in human breast cancer 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
Free Radical Biology Medicine 30(3)260-267 2001
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Up-regulated Genes in MCF-7 Cells Overexpressing
MnSOD
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Down-regulated Genes in MCF-7 Cells
Overexpressing MnSOD
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Which genes or proteins regulated by MnSOD are
important? HIF-1 may be crucial because it
controls tumor proliferation and angiogenesis
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Hypoxic (1 O2) Accumulation of HIF-1? Protein
is Suppressed by Plasmid Transfection of MnSOD

Clones
MnSOD Activity
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Increased MnSOD Activity by Adenovirus Infection
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Hypoxic (1 O2) Accumulation of HIF-1? Protein is
Suppressed by Adenoviral Transduction of MnSOD
AdMnSOD MOI
Hela
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Regression Lines Showing a Biphasic Effect
HIF-1?/tubulin
Relative MnSOD activity (-fold)
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Vascular Endothelial Growth Factor (VEGF)
VEGF is an essential factor mediating new
blood vessel formation and angiogenesis. Inject
ion of VEGF into rat skin induces
angiogenesis. VEGF gene expression is
regulated by HIF-1 during hypoxia.
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VEGF Production Increased When Cells Were
Exposed to Hypoxia
VEGF (pg/106 cells)
time in hypoxia (h)
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MnSOD Suppressed Hypoxic Induction of VEGF in
MCF-7 Cells
VEGF (pg/106 cells)
time in hypoxia (h)
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Conclusions

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

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Conclusions

• MnSOD levels are low in cancer cells due to a
  variety of reasons.
• Overexpression of MnSOD inhibits cancer cell
  growth both in vitro and in vivo.
• The growth inhibitory effects of MnSOD
  over-expression in human glioma cells are mainly
  due to hydrogen peroxide.
• MnSOD overexpression inhibits cell growth due to
  its effects on signal transduction.

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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.

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ANTITUMOR THERAPIES BASED ON ANTIOXIDANT
MODULATION

• A vision for the future

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Reactive Oxygen Species and Antioxidant Schematic
Diagram
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Mechanism of H2O2 Increase with BCNU and AT
Addition
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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
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AdMnSOD Plus BCNU Decreased Oral Cancer Growth In
Vivo
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Conclusion

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

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Thank you!