The Mechanism of the Tumor Suppressive Effects of MnSOD Overexpression

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 Location MnSOD CuZnSOD 88,000 32,000 Mitochondria Cytosol, nucleus Extracellular Extracellular ECSOD ECMnSOD 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 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. Clones transfected with Ile58 MnSOD cDNA. The clones were named Mn1, Mn11, Mn28, Mn40, Mn44, Mn52, Mn59, and Mn63. Mn 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 59 Mn 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 9 3 5 2 4 1 9 3 9 9 20 19 7 8 33 31 7 6 5 6 8 7 8 7 1 1 1 1 40 R elativ e M n S O D A ctiv ity (fo ld ) 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 Relative IDV (fold) 30 40 250 200 Tumor Volume (mm3) 150 100 WT Neo4 SOD15 SOD18 SOD23 SOD50 Mn1 Mn28 Mn40 Mn59 WT Neo4 SOD15 SOD18 SOD23 SOD50 Mn1 Mn11 Mn28 4/4 3/4 1/4 3/4 2/4 1/4 1/4 0/4 1/4 Mn40 2/4 0/4 50 Mn44 Mn52 Mn59 0/4 1/4 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mn63 0/4 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 V1 V2 Total SOD (U/mg protein) Antioxidant Enzyme Activity in Parental, MnSOD (U/mg protein) CuZnSOD (U/mg protein) Catalase (k/mg protein) GPx (mU/mg protein) 47  6 43  5 54  4 31  5 28  2 33  4 16  8 15  5 21 5 106  10 115  12 102  10 11  2 91 10  1 Mn1 Mn2 Mn3 Mn4 Mn5 Mn6 80  5 112  9 74  5 136  6 85  5 184  9 64  2* 95  6* 62  6* 116  4* 71  8* 158  7* 16  5 17  10 13  8 20  7 14  8 26  12 119  5 112  5 98  8 106  6 102  7 114  10 18  2* 21  3* 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 Human glioma U118-9 cells SOD2 Neo Zeo35 A MnSOD over-expressing transfectant A vector control for SOD2 A vector control for MnSOD-GPX double transfectants S-GPXs U118  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 4/7 Neo 62.5 5/8 SOD 2 25 2/8 Zeo3 5 37.5 3/8 S– GPX70 25 2/8 S– GPX86 75 6/8 S– GPX146 85.7 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 D13866 M23410 Gene name MET Growth arrest and DNA-damageinducible protein (GADD153) CD9 A-catenin (cadherin-associated protein) Plakoglobin (desmoplakin III) Function oncogene SOD/MC F (Fold*) 42 DNA damage  33 response cell adhesion cell-cell interaction cell-cell interaction  7  2 14 *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 1% O2 HIF-1 AdMnSOD MOI 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 hyp. medium no-hyp. medium 2500 2000 1500 1000 500 0 0 4 8 12 16 20 24 28 hypoxia lysate VEGF (pg/106 cells) 3000 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 -G lu ta m a te  -G C S BSO  - G lu ta m y l-c y s te in e GS H 2O BCNU NADPH GSH GPX H 2O 2 SOD O2 - 6 -P h o s p h o g lu c o n o la c to n e G -6 -P D GR GSSG NADP- C AT O 2 + H 2O AT G lu c o s e 6 -p h o s p h a te DHEA D e o x y g lu c o s e G lu c o s e Mechanism of H2O2 Increase with BCNU and AT Addition O2 - H 2O 2 GPx H 2O 2 BCNU c a ta la s e H 2O 2 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 **p<0.005 vs. BCNU 48 ** ** 40 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!