Synergistic induction of heme oxygenase-1 by the components of an antioxi- dant supplement Protandim Kalpana Velmurugan, Jawed Alam, Joe M. McCord, Subbiah Pugazhen- thi PII: S0891-5849(08)00673-4 DOI: doi: 10.1016/j.freeradbiomed.2008.10.050 Reference: FRB 9615 To appear in: Free Radical Biology and Medicine Received date: 8 July 2008 Revised date: 23 September 2008 Accepted date: 31 October 2008 Please cite this article as: Kalpana Velmurugan, Jawed Alam, Joe M. McCord, Sub- biah Pugazhenthi, Synergistic induction of heme oxygenase-1 by the components of an antioxidant supplement Protandim, Free Radical Biology and Medicine (2008), doi: 10.1016/j.freeradbiomed.2008.10.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Synergistic induction of heme oxygenase-1 by the components of an antioxi-dant supplement Protandim
Kalpana Velmurugan, Jawed Alam, Joe M. McCord, Subbiah Pugazhen-thi
Received date: 8 July 2008Revised date: 23 September 2008Accepted date: 31 October 2008
Please cite this article as: Kalpana Velmurugan, Jawed Alam, Joe M. McCord, Sub-biah Pugazhenthi, Synergistic induction of heme oxygenase-1 by the components ofan antioxidant supplement Protandim, Free Radical Biology and Medicine (2008), doi:10.1016/j.freeradbiomed.2008.10.050
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
ACCEPTED MANUSCRIPT Synergistic induction of Heme oxygenase-1
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Synergistic induction of heme oxygenase-1 by the components of an antioxidant supplement Protandim
Kalpana Velmurugan1, Jawed Alam2, Joe M. McCord3 and Subbiah Pugazhenthi1
1Division of Endocrinology, 3Division of Pulmonary Sciences, Department of Medicine, University of Colorado Denver, Aurora, CO and
1Section of Endocrinology, Veterans Affairs Medical Center, Denver, CO; 2Department of Molecular Genetics, Ochsner Medical Center, New Orleans, LA
ACKNOWLEDGEMENTS: This work was supported by grants from Juvenile diabetes Research Foundation (5-2005-1104, to S.P.), American Diabetes Association (1-06-JF-40, to S.P.) and LifeVantage Corp. (to S.P. and J.M.M.). We thank the support provided
by Microscopy Core facility at Denver VA Medical Center.
Address for correspondence Subbiah Pugazhenthi, Ph.D. Division of Endocrinology, Department of Medicine,
University of Colorado Denver P.O. Box 6511; Mail Stop 8106,
Protandim. The results are mean of four independent observations. * P< 0.001 vs untreated
control. #P<0.01 and **P<0.001 vs Protandim at corresponding doses.
Figure 5. ARE site- and Nrf2-dependent induction of HO-1 promoter by Protandim:
A: The plasmids ∆ (E1) and ∆ (E2) were obtained by deletion of 600-base pair
(SacI/SacI) fragment and 161-base pair AflII/BsrBI fragment respectively from the 15-kb
promoter fragment of mouse ho-1 gene, and were cloned into luciferase reporter gene.
SK-N-MC cells cultured in 12 well dishes to 70% confluence were transfected with the
indicated HO-1 promoter constructs linked to firefly luciferase along with constitutively
active renilla luciferase using LipofectAMINE 2000 reagent. After 6 h of transfection, the
cells were exposed to 20 µg/ml of Protandim for another 18 h. Cell lysates were
prepared and luciferase activities were measured. The ratios of activities of firefly
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ACCEPTED MANUSCRIPT Synergistic induction of Heme oxygenase-1
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luciferase and renilla luciferase were determined. The results are mean ± SE of four
independent observations. * P< 0.001 vs untreated control; # P < 0.01; **P<0.001 with
respect to full length promoter activation by Protandim. B: SK-N-MC cells were
transfected with either (1) full length promoter of HO-1 linked to firefly luciferase reporter
and vector (pEF); or (2) the promoter/reporter construct plus an expression construct for
Keap1 or 3) the promoter/reporter construct plus a dominant negative Nrf2 expression
construct. After 6 h of transfection, the cells were exposed to 20 µg/ml of Protandim for
18 h. Cell lysates were prepared for the assay of luciferase activities. The results are
mean ± SE of four independent experiments. * P< 0.001 vs untreated control; # P <
0.001 with respect to vector control.
Figure 6. Nuclear translocation of Nrf2 by Protandim: A: SK-N-MC cells cultured on
cover slips were exposed to 20 µg/ml of Protandim. After 6 h, cells were fixed in 4%
paraformaldehyde, permeabilized and immunostained for active Nrf2 (Cy3; red). The
nuclei were stained with DAPI (blue). Images were examined by fluorescent
microscopy. The merge of Cy3 and DAPI is shown as overlay. The images presented
here are representative of multiple fields from three independent experiments.
Figure 7. Role of multiple signaling pathways on induction of HO-1 expression by
Protandim: SK-N-MC cells were preincubated in the presence of 250 nM of Akt
inhibitor IV, 30 µM of LY294002, 1 µM of rottlerin, 10 µM of U0126 or 20 µM of
SB203580 for 20 µM of SP600125 for 30 min followed by exposure to 20 µg/ml of
Protandim for 24 h. A: Cell lysates were electrophoresed and immunoblotted for HO-1.
The blots were then reprobed with the antibody for β Actin. A representative of four blots
is presented for each inhibitor. B: The intensities of bands were quantified by
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densitometry using Fluor-S MultiImager and Quantity One software from Bio-Rad. HO-1
levels were corrected for β Actin expression. * P<0.001 when compared to untreated
control. # P<0.001 with respect to Protandim-treated cells in the absence of inhibitors.
Figure 8: Protandim-mediated increase in cellular total glutathione content: MIN6
(A) and SK-N-MC cells (B) cultured in 100 mm dishes to 70% confluence were exposed
to indicated concentrations of Protandim for 24 h. Cell lysates were prepared for the
assay of total glutathione. * P<0.001 when compared to untreated control.
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References:
[1] Robertson, R. P. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem 279:42351-4; 2004.
[2] Rottkamp, C. A.; Nunomura, A.; Raina, A. K.; Sayre, L. M.; Perry, G.; Smith, M. A. Oxidative stress, antioxidants, and Alzheimer disease. Alzheimer Dis Assoc Disord 14 Suppl 1:S62-6; 2000.
[3] Harrison, D.; Griendling, K. K.; Landmesser, U.; Hornig, B.; Drexler, H. Role of oxidative stress in atherosclerosis. Am J Cardiol 91:7A-11A; 2003.
[4] Moreira, P. I.; Smith, M. A.; Zhu, X.; Honda, K.; Lee, H. G.; Aliev, G.; Perry, G. Oxidative damage and Alzheimer's disease: are antioxidant therapies useful? Drug News Perspect 18:13-9; 2005.
[5] Bjelakovic, G.; Nikolova, D.; Gluud, L. L.; Simonetti, R. G.; Gluud, C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. Jama 297:842-57; 2007.
[6] Podmore, I. D.; Griffiths, H. R.; Herbert, K. E.; Mistry, N.; Mistry, P.; Lunec, J. Vitamin C exhibits pro-oxidant properties. Nature 392:559; 1998.
[7] Wu, L.; Noyan Ashraf, M. H.; Facci, M.; Wang, R.; Paterson, P. G.; Ferrie, A.; Juurlink, B. H. Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system. Proc Natl Acad Sci U S A 101:7094-9; 2004.
[8] Juurlink, B. H. Therapeutic potential of dietary phase 2 enzyme inducers in ameliorating diseases that have an underlying inflammatory component. Can J Physiol Pharmacol 79:266-82; 2001.
[9] Joe, B.; Vijaykumar, M.; Lokesh, B. R. Biological properties of curcumin-cellular and molecular mechanisms of action. Crit Rev Food Sci Nutr 44:97-111; 2004.
[10] Kishore, K.; Singh, M. Effect of bacosides, alcoholic extract of Bacopa monniera Linn. (brahmi), on experimental amnesia in mice. Indian J Exp Biol 43:640-5; 2005.
[11] Mandel, S.; Weinreb, O.; Amit, T.; Youdim, M. B. Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases. J Neurochem 88:1555-69; 2004.
[12] Nelson, S. K.; Bose, S. K.; Grunwald, G. K.; Myhill, P.; McCord, J. M. The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy. Free Radic Biol Med 40:341-7; 2006.
[13] Pugazhenthi, S.; Akhov, L.; Selvaraj, G.; Wang, M.; Alam, J. Regulation of Heme oxygenase-1 expression by demethoxy curcuminoids through Nrf2 by a PI 3-kinase/Akt-mediated pathway in mouse beta cells. Am J Physiol Endocrinol Metab; 2007.
[14] Wu, C. C.; Hsu, M. C.; Hsieh, C. W.; Lin, J. B.; Lai, P. H.; Wung, B. S. Upregulation of heme oxygenase-1 by Epigallocatechin-3-gallate via the phosphatidylinositol 3-kinase/Akt and ERK pathways. Life Sci 78:2889-97; 2006.
[15] Ryter, S. W.; Alam, J.; Choi, A. M. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 86:583-650; 2006.
[16] Alam, J.; Wicks, C.; Stewart, D.; Gong, P.; Touchard, C.; Otterbein, S.; Choi, A. M.; Burow, M. E.; Tou, J. Mechanism of heme oxygenase-1 gene activation by cadmium in
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT Synergistic induction of Heme oxygenase-1
28
MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. J Biol Chem 275:27694-702; 2000.
[17] Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72:248-254; 1976.
[18] Anderson, M. E. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548-55; 1985.
[19] Tiedge, M.; Lortz, S.; Drinkgern, J.; Lenzen, S. Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46:1733-42; 1997.
[20] Alam, J.; Stewart, D.; Touchard, C.; Boinapally, S.; Choi, A. M.; Cook, J. L. Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 274:26071-8; 1999.
[21] Gong, P.; Stewart, D.; Hu, B.; Vinson, C.; Alam, J. Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite. Arch Biochem Biophys 405:265-74; 2002.
[22] Hock, T. D.; Liby, K.; Wright, M.; McConnell, S.; Schorpp-Kistner, M.; Ryan, T. M.; Agarwal, A. JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells. J Biol Chem; 2007.
[23] Itoh, K.; Tong, K. I.; Yamamoto, M. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med 36:1208-13; 2004.
[24] Martin, D.; Rojo, A. I.; Salinas, M.; Diaz, R.; Gallardo, G.; Alam, J.; De Galarreta, C. M.; Cuadrado, A. Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem 279:8919-29; 2004.
[25] Kronke, G.; Bochkov, V. N.; Huber, J.; Gruber, F.; Bluml, S.; Furnkranz, A.; Kadl, A.; Binder, B. R.; Leitinger, N. Oxidized phospholipids induce expression of human heme oxygenase-1 involving activation of cAMP-responsive element-binding protein. J Biol Chem 278:51006-14; 2003.
[26] Wu, C. C.; Hsieh, C. W.; Lai, P. H.; Lin, J. B.; Liu, Y. C.; Wung, B. S. Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein. Toxicol Appl Pharmacol 214:244-52; 2006.
[27] Prestera, T.; Holtzclaw, W. D.; Zhang, Y.; Talalay, P. Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc Natl Acad Sci U S A 90:2965-9; 1993.
[28] Stocker, R.; Perrella, M. A. Heme oxygenase-1: a novel drug target for atherosclerotic diseases? Circulation 114:2178-89; 2006.
[29] Deshane, J.; Wright, M.; Agarwal, A. Heme oxygenase-1 expression in disease states. Acta Biochim Pol 52:273-84; 2005.
[30] Pileggi, A.; Molano, R. D.; Berney, T.; Cattan, P.; Vizzardelli, C.; Oliver, R.; Fraker, C.; Ricordi, C.; Pastori, R. L.; Bach, F. H.; Inverardi, L. Heme oxygenase-1 induction in islet cells results in protection from apoptosis and improved in vivo function after transplantation. Diabetes 50:1983-91; 2001.
[31] Tobiasch, E.; Gunther, L.; Bach, F. H. Heme oxygenase-1 protects pancreatic beta cells from apoptosis caused by various stimuli. J Investig Med 49:566-71; 2001.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT Synergistic induction of Heme oxygenase-1
29
[32] Ribeiro, M. M.; Klein, D.; Pileggi, A.; Molano, R. D.; Fraker, C.; Ricordi, C.; Inverardi, L.; Pastori, R. L. Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic beta-cells. Biochem Biophys Res Commun 305:876-81; 2003.
[33] Li, Y.; Li, G.; Dong, W.; Chen, J.; Lu, D.; Tan, J. Transplantation of rat islets transduced with human heme oxygenase-1 gene using adenovirus vector. Pancreas 33:280-6; 2006.
[34] Behl, C. Amyloid beta protein toxicity and oxidative stress in Alzheimer's disease. Cell Tissue Res 290:471-480; 1997.
[35] Nicolle, M. M.; Gonzalez, J.; Sugaya, K.; Baskerville, K. A.; Bryan, D.; Lund, K.; Gallagher, M.; McKinney, M. Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents. Neuroscience 107:415-31; 2001.
[36] Markesbery, W. R. Oxidative stress hypothesis in Alzheimer's disease. Free Radical Biology & Medicine 23:134-147; 1997.
[37] Smith, M. A.; Rottkamp, C. A.; Nunomura, A.; Raina, A. K. Oxidative stress in Alzheimer's disease. Biochim Biophys Acta 1502:139-144; 2000.
[38] Keller, J. N.; Schmitt, F. A.; Scheff, S. W.; Ding, Q.; Chen, Q.; Butterfield, D. A.; Markesbery, W. R. Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 64:1152-6; 2005.
[39] Hensley, K.; Hall, N.; Subramaniam, R.; Cole, P.; Harris, M.; Aksenov, M.; Aksenova, M.; Gabbita, S. P.; Wu, J. F.; Carney, J. M.; et al. Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation. J Neurochem 65:2146-56; 1995.
[40] Yao, Y.; Chinnici, C.; Tang, H.; Trojanowski, J. Q.; Lee, V. M.; Pratico, D. Brain inflammation and oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis. J Neuroinflammation 1:21; 2004.
[41] Darville, M. I.; Eizirik, D. L. Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells. Diabetologia 41:1101-8; 1998.
[42] Robertson, R. P.; Harmon, J. S. Diabetes, glucose toxicity, and oxidative stress: A case of double jeopardy for the pancreatic islet beta cell. Free Radic Biol Med 41:177-84; 2006.
[43] Emamaullee, J. A.; Shapiro, A. M. Interventional strategies to prevent beta-cell apoptosis in islet transplantation. Diabetes 55:1907-14; 2006.
[44] Bertera, S.; Crawford, M. L.; Alexander, A. M.; Papworth, G. D.; Watkins, S. C.; Robbins, P. D.; Trucco, M. Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes. Diabetes 52:387-93; 2003.
[45] Robertson, R. P.; Harmon, J. S. Pancreatic islet beta-cell and oxidative stress: the importance of glutathione peroxidase. FEBS Lett 581:3743-8; 2007.
[46] Ahmad, M.; Saleem, S.; Ahmad, A. S.; Ansari, M. A.; Yousuf, S.; Hoda, M. N.; Islam, F. Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol 24:137-47; 2005.
[47] Dhanasekaran, M.; Tharakan, B.; Holcomb, L. A.; Hitt, A. R.; Young, K. A.; Manyam, B. V. Neuroprotective mechanisms of ayurvedic antidementia botanical Bacopa monniera. Phytother Res 21:965-9; 2007.
[48] Jyoti, A.; Sharma, D. Neuroprotective role of Bacopa monniera extract against aluminium-induced oxidative stress in the hippocampus of rat brain. Neurotoxicology 27:451-7; 2006.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT Synergistic induction of Heme oxygenase-1
30
[49] Russo, A.; Izzo, A. A.; Borrelli, F.; Renis, M.; Vanella, A. Free radical scavenging capacity and protective effect of Bacopa monniera L. on DNA damage. Phytother Res 17:870-5; 2003.
[50] Pawar, R. S.; Khan, S. I.; Khan, I. A. Glycosides of 20-deoxy derivatives of jujubogenin and pseudojujubogenin from Bacopa monniera. Planta Med 73:380-3; 2007.
[51] Papaiahgari, S.; Kleeberger, S. R.; Cho, H. Y.; Kalvakolanu, D. V.; Reddy, S. P. NADPH oxidase and ERK signaling regulates hyperoxia-induced Nrf2-ARE transcriptional response in pulmonary epithelial cells. J Biol Chem 279:42302-12; 2004.
[52] Huang, H. C.; Nguyen, T.; Pickett, C. B. Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci U S A 97:12475-80; 2000.
[53] Erickson, A. M.; Nevarea, Z.; Gipp, J. J.; Mulcahy, R. T. Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence. J Biol Chem 277:30730-7; 2002.
[54] Mulcahy, R. T.; Wartman, M. A.; Bailey, H. H.; Gipp, J. J. Constitutive and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase heavy subunit gene is regulated by a distal antioxidant response element/TRE sequence. J Biol Chem 272:7445-54; 1997.
[55] Wu, G.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D. Glutathione metabolism and its implications for health. J Nutr 134:489-92; 2004.
[56] Lotito, S. B.; Frei, B. Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic Biol Med 41:1727-46; 2006.
[57] Halliwell, B.; Rafter, J.; Jenner, A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am J Clin Nutr 81:268S-276S; 2005.
[58] Garodia, P.; Ichikawa, H.; Malani, N.; Sethi, G.; Aggarwal, B. B. From ancient medicine to modern medicine: ayurvedic concepts of health and their role in inflammation and cancer. J Soc Integr Oncol 5:25-37; 2007.