Anti-apoptotic Activity of Heme Oxygenase-1 Up-regulated by Etoposide in Human Retinal Pigment Epithelial Cells

Etoposide에 의한 인간 망막색소상피세포인 ARPE-19 세포의 아폽토시스 과정에서 Heme oxygenase-1의 항아폽토시스 기능에 대한 연구

  • Lee, Sang-Kwon (Department of Cardiovascular Surgery and Medical Research Institute, Pusan National University School of Medicine) ;
  • Song, Ju-Dong (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Kim, Kang-Mi (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Kim, Jong-Min (Department of Anatomy and Cell Biology, Dong-A University School of Medicine) ;
  • Lee, Sang-Yull (Department of Biochemistry, Pusan National University School of Medicine) ;
  • Yoo, Young-Hyun (Department of Anatomy and Cell Biology, Dong-A University School of Medicine) ;
  • Park, Young-Chul (Department of Microbiology & Immunology, Pusan National University School of Medicine)
  • 이상권 (부산대학교 의학전문대학원 흉부외과학교실 및 의학연구소) ;
  • 송주동 (부산대학교 의학전문대학원 미생물학 및 면역학교실) ;
  • 김강미 (부산대학교 의학전문대학원 미생물학 및 면역학교실) ;
  • 김종민 (동아대학교 의과대학 해부학교실) ;
  • 이상률 (부산대학교 의학전문대학원 생화학교실) ;
  • 유영현 (동아대학교 의과대학 해부학교실) ;
  • 박영철 (부산대학교 의학전문대학원 미생물학 및 면역학교실)
  • Published : 2007.09.30


The topoisomerase II inhibitor etoposide causes an accumulation of DNA double strand breaks within the nuclei of cells. In this study, we investigated the effect of etoposide on the cell growth and apoptosis of human RPE cells. Etoposide evoked a significant inhibition of cell growth, and also induced DNA fragmentation in ARPE-19 cells. In addition, etoposide significantly up-regulated the expression of heme oxygenase-1 (HO-1), which is a stress-responsive protein and is known to play a protective role against the oxidative injury. And, etoposide-induced HO-1 expression was affected by the ROS scavenger N-acetyl cysteine. We also used oligonucleotides interfering with HO-1 mRNA (siRNA) for the inhibition of HO-1 expression. Interestingly, knock-down of the HO-1 gene significantly increased the level of DNA fragmentation in etoposide-treated ARPE-19 cells. In conclusion, these results suggest that up-regulated HO-1 plays as an anti-apoptotic factor in the process of apoptosis of ARPE-19 cells stimulated by etoposide.


  1. Alizadeh, M., M. Wada, C. M. Celfman, J. T. Handa and L. M. Hjelmeland. 2001. Downregulation of differentiation specific gene expression by oxidative stress in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 42, 2706-2713
  2. Angkeow, P., S. S. Deshpande, B. Qi, Y. X. Liu, Y. C. Park, B. H. Jeon, M. Ozaki and K. Irani. 2002. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ. 9, 717-725
  3. Applegate, L. A, P. Luscher and R. M. Tyrrell. 1991. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 51, 974-978
  4. Bok, D. 1993. The retinal pigment epithelium: a versatile partner in vision. J. Cell Sci. Suppl. 17, 189-195
  5. Burden, D. A and N. Osheroff. 1998. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochem. Biophys. Acta 1400, 139-154
  6. Choi, B. M., H. O. Pae, Y. R. Jeong, G. S. Oh, C. D. Jun, B. R. Kim, Y. M. Kim and H. T. Chung. 2004. Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to Fas-mediated apoptosis: involvement of iron released by HO-1. Free Radic. Biol. Med. 36, 858-871
  7. Detrick, B., D. A. Newsome, C. M. Percopo and J. J. Hooks. 1985. Class II antigen expression and gamma interferon modulation of monocytes and retinal pigment epithelial cells from patients with retinitis pigmentosa. Clin. Immunol. Immunopathol. 36, 201-211
  8. Duckers, H. J., M. Boehm, A. L. True, S. F. Yet, H. San, J. L. Park, W. R. Clinton, M. E. Lee, G. J. Nabel and E. G. Nabel. 2001. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat. Med. 7, 693-698
  9. Elner, S. G., R. M. Strieter, V. M. Elner, B. J. Rollins, M. A. Del Monte and S. L. Kunkel. 1991. Monocyte chemotactic protein gene expression by cytokine-treated human retinal pigment epithelial cells. Lab Invest. 64, 819-825
  10. Elner, S. G., V. M. Elner, M. A. Pavilack, R. F. 3rd Todd, L. Mayo-Bond, W. A. Franklin, R. M. Strieter, S. L. Kunkel and A. R. Huber. 1992. Modulation and function of intercellular adhesion molecule-1 (CD54) on human retinal pigment epithelial cells. Lab Invest. 66, 200-211
  11. Elner, V. M., R. M. Strieter, S. G. Elner, M. Baggiolini, I. Lindley and S. L. Kunkel. 1990. Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigment epithelial cells. Am. J. Pathol. 136, 745-750
  12. Gao, H. and J. G. Hollyfield. 1992. Aging of the human retina. Differential loss of neurons and retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 33, 1-17
  13. Griffith, T. S., T. Brunner, S. M. Fletcher, D. R. Green and T. A. Ferguson. 1995. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 17, 1189-1192
  14. Hande, K. R. 1998. Etoposide: four decades of development of a topoisomerase II inhibitor. Eur. J. Cancer 34, 1514-1521
  15. Honda, S., L. M. Hjelmeland and J. T. Handa. 2000. The use of hyperoxia to induce chronic mild oxidative stress in RPE cells in vitro. Mol. Vis. 7, 63-70
  16. Hueber, A, P. Esser, K. Heimann, N. Kociok, S. Winter and M. Weller. 1998. The topoisomerase I inhibitors, camptothecin and beta-Iapachone, induce apoptosis of human retinal pigment epithelial cells. Exp. Eye Res. 67, 525-530
  17. Hunt, R. C., D. M. Hunt, N. Gaur and A. Smith. 1996. Hemopexin in the human retina: protection of the retina against heme-mediated toxicity. J. Cell Physiol. 168, 71-80<71::AID-JCP9>3.0.CO;2-5
  18. Immenshuh, S. and G. Ramadori. 2000. Gene regulation of heme oxygenase-1 as a therapeutic target. Biochem. Pharmacol. 60, 1121-1128
  19. Jaffe, G. J., Le. L. Van, F. Valea, S. Haskill, W. Roberts, W. P. Arend, A. Stuart and W. P. Peters. 1992. Expression of interleukin-1 alpha, interleukin-1 beta, and an interleukin-1 receptor antagonist in human retinal pigment epithelial cells. Exp. Eye Res. 55, 325-335
  20. Jaffe, G. J., W. L. Roberts, H. L. Wong, A. D. Yurochko and C. J. Cianciolo. 1995. Monocyte-induced cytokine expression in cultured human retinal pigment epithelial cells. Exp. Eye Res. 60, 533-543
  21. Kutty, R. K., G. Kutty, B. Wiggert, C. J. Chader, R. M. Darrow and D. T. Organisciak. 1995. Induction of heme oxygenase 1 in the retina by intense visible light: suppression by the antioxidant dimethylthiourea. Proc. Natl. Acad. Sci. USA 92, 1177-1181
  22. Maines, M. D. 1997. The heme oxygenase system: a regulator of second messenger gases. Annu. Rev. Pharmacol. Taxicol. 37, 517-554
  23. Mizumoto, K., R. J. Rothman and J. L. Farber. 1994. Programmed cell death (apoptosis) of mouse fibroblasts is induced by the topoisomerase II inhibitor etoposide. Mol. Pharmacol. 46, 890-895
  24. Otterbein, L. E. and A. M. K. Choi. 2000. Heme oxygenase: colors of defense against cellular stress. Am. J. Physiol. 279, L1029-L1037
  25. Park, S. E., J. D. Song, K. M. Kim, Y. M. Park, Y. H. Yoo and Y. C. Park. 2007. Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells. FEBS Lett. 581, 180-186
  26. Planck, S. R., X. N. Huang, J. E. Robertson and J. T. Rosenbaum. 1993. Retinal pigment epithelial cells produce interleukin-1 beta and granulocyte-macrophage colony-stimulating factor in response to interleukin-1 alpha. Curr. Eye Res. 12, 205-212
  27. Platts, K. E., M. T. Benson, I. G. Rennie, R. M. Sharrard and R. C. Rees. 1995. Cytokine modulation of adhesion molecule expression on human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 36, 2262-2269
  28. Poss, K. D. and S. Tonegawa. 1997. Reduced stress defense in heme oxygenase-1-deficient cells. Proc. Natl. Acad. Sci. USA 94, 10925-10930
  29. Schwartzman, M. L., J. Masferrer, M. W. Dunn, J. C. McGiff and N. G. Abraham. 1987. Cytochrome P450, drug metabolizing enzymes and arachidonic acid metabolism in bovine ocular tissues. Curr. Eye Res. 6, 623-630
  30. Steinhorst, U. H., E. P. Chen, D. L. Hatchell, C. P. Samsa, P. Y. Saloupis, J. Westendorf and R. Machemer. 1993. Aclacinomycin A in the treatment of experimental proliferative vitreoretinopathy. Efficacy and toxicity in the rabbit eye. Invest. Ophthalmol. Vis. Sci. 34, 1753-1760
  31. Tate, D. J., D. A. Newsome and P. D. Oliver. 1993. Metallothionein shows an age-related decrease in human macular retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 34, 2348-2351
  32. Tate, D. J., M. V. Miceli and D. A. Newsome. 1995. Phagocytosis and hydrogen peroxide induce catalase and metallothionein gene expression in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 36, 1271-1279
  33. Varma, S. D. and R. D. Richards. 1988. Visual disorders and oxidative reactions. pp. 71-88, In Chow, C. K. (eds), Cellular antioxidant defense mechanisms, CRC Press, Boca Raton
  34. Velikay, M., U. Stolba, A. Wedrich, P. Datlinger, J. Akramian and S. Binder. 1994. The antiproliferative effect of fractionized radiation therapy: optimization of dosage. Doc. Ophthalmol. 87, 265-269
  35. Wiegund, R. D., J. G. Jose, L. M. Rapp and R. E. Anderson. 1984. Free radicals and damage to ocular tissues. pp. 317-353, In Armstrong, D., R. S. Sohal, R. C. Cutler and T. F. Slater (eds), Free radicals in molecular biology, aging, and disease, Raven Press, New York
  36. Wu, W. C, Y. H. Kao and D. N. Hu. 2002. A comparative study of effects of antiproliferative drugs on human retinal pigment epithelial cells in vitro. J. Ocul. Pharmacol. Ther. 18, 251-264
  37. Yoon, H. S., J. H. Rho, K. W. Yoo, W. C. Park, S. H. Rho, Y. H. Choi, H. Suh, N. D. Kim, K. S. Yoo and Y. H. Yoo. 2001. Synthetic bile acid derivatives induce nonapoptotic death of human retinal pigment epithelial cells. Curr. Eye Res. 22, 367-374
  38. Yoon, H. S., S. C. Moon, N. D. Kim, B. S. Park and Y. H. Yoo. 2000. Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP. Biochem. Biophys. Res. Commun. 276, 151-156
  39. Zinn, K. and J. V. Benjamin-Henkind. 1979. Anatomy of the human retinal pigment epithelium, pp. 3-31, In Zinn, K. M. and M. Marmor (eds), The retinal pigment epithelium, Harvard University Press, Cambridge
  40. Liles, M. R., D. A. Newsome and P. D. Oliver. 1991. Antioxidant enzymes in the aging human retinal pigment epithelium. Arch. Ophthal. 109, 1285-1288
  41. Planck, S. R., T. T. Dang, D. Craves, D. Tara, J. C. Ansel and J. T. Rosenbaum. 1992. Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1. Invest. Ophthalmol. Vis. Sci. 33, 78-82