Tuberculosis and Respiratory Diseases

The Korean Academy of Tuberculosis and Respiratory Diseases (대한결핵및호흡기학회)
권호 표지

Characterization of Cigarette Smoke Extract (CSE)-induced Cell Death in Lung Epithelial Cells

폐상피세포에서 흡연추출물-유도성 세포사에 관한 연구

  • Choi, Eun Kyung (Department of Internal Medicine, Dankook University College of Medicine) ;
  • Kim, Yun Seup (Department of Internal Medicine, Dankook University College of Medicine) ;
  • Park, Jae Seuk (Department of Internal Medicine, Dankook University College of Medicine) ;
  • Jee, Young Koo (Department of Internal Medicine, Dankook University College of Medicine) ;
  • Lee, Kye Young (Department of Internal Medicine, Dankook University College of Medicine)
  • 최은경 (단국대학교 의과대학 내과학교실) ;
  • 김윤섭 (단국대학교 의과대학 내과학교실) ;
  • 박재석 (단국대학교 의과대학 내과학교실) ;
  • 지영구 (단국대학교 의과대학 내과학교실) ;
  • 이계영 (단국대학교 의과대학 내과학교실)
  • Received : 2004.08.30
  • Accepted : 2004.12.07
  • Published : 2005.01.30
Download PDF
⟨ Previous Next ⟩

Abstract

Emphysema is characterized by air space enlargement and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was performed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cytochrome c release was confirmed by using immunofluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near completely blocked by N-acetylcystein and bcl-2 overexpression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the pathogenesis of emphysema.

배 경 : 폐기종에서 발생하는 폐포 파괴의 원인으로서 전통적으로 protease/anti-protease 불균형과 산화성 스트레스가 주요 가설로 여겨져 왔으나 최근 폐포세포의 아포프토시스가 폐포파괴 및 폐기종의 원인이 된다는 이론이 제기되고 있어서 A549 폐상피세포에서 흡연추출물에 의한 세포사의 특성을 규명하고자 본 연구를 시행하였다. 방 법 : A549 폐상피세포주에서 여러 농도의 흡연추출물 및 억제제를 첨가한 후 MTT assay를 잉용하여 세포생존율을 측정하였다. 세포사 분석은 FACScan을 이용한 DNA 분절확인, 전자현미경 검사, Hoecst/PI 이중염색을 이용한 현광현미경 검사를 이용하였고 cyt-ochrome c 유리는 면역형광법을 이용하였다. Bcl-2 과발현세포주를 이용하여 bcl-2의 역할을 확인하였고 p53 Western blot 및 HPV-E6 과발현 세포주를 이용하여 p53의 역할을 확인하였다. 결 과 : A549 세포주에서 흡연추출물에 의한 세포사는 FACScan에서 DNA 분절에 의한 subG1 분획의 확인 및 Hoecst/PI 이중염색 및 현광현미경 소견 상 아포프토시스임이 확인되었고 전자현미경 소견상 저농도에서는 아포프토시스가 발생하지만 고농도에서는 괴사가 발생함을 확인하였다. Cytochrome c가 세포질로 유리됨을 확인하였으나 caspase 억제제에 의해서 세포사가 차단되지 않았다. 흡연추출물에 의한 세포사는 Bcl-2과발현에 의해 억제되었고 p53활성화를 유도하고 p53이 기능적으로 knock-out 된 세포주에서 억제되었다. 결 론 : 흡연추출물에 의한 폐상피세포의 세포사는 저농도에서는 아포프토시스를 고농도에서는 괴사를 유도하고 bcl-2 및 p53 경로가 중요한 역할을 담당하며 이는 폐기종 발생기전에 있어서 폐세포 세포사 이론을 뒷바침하는 자료로 활용될 수 있다고 생각된다.

Keywords

References

  1. Holbrrok JH, Groundy SM, Hennekens CH, Kannel WB, Strong JP. Cigarette smoking and cardiovascular diseases: a statement for health professionals by a task force appointed by the steering committee of the American Heart Association. Circulation 1984;70:1114A-7A
  2. Altshuler B. Quantitative models for lung cancer induced by cigarette smoke. Environ Health Perspect 1989;81:107 https://doi.org/10.2307/3430814
  3. Higgins M Epidemiology of COPD: state of art. Chest 1984;85:3S-8S
  4. Sethi JM, Rochester CL. Smoking and chronic obstructive pulmonary disease. Clin Chest Med 2000;21:67-86 https://doi.org/10.1016/S0272-5231(05)70008-3
  5. Rahman I, MacNee W. Role of oxidants/antioxidants in smoking-induced lung disease. Free Radic Biol Med 1996;21:669-81 https://doi.org/10.1016/0891-5849(96)00155-4
  6. Stockely RA. Proteases/antiproteases: pathogenesis and role in therapy. Clin Pulm Med 1998;5:203-10 https://doi.org/10.1097/00045413-199807000-00001
  7. Church T, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspect 1985;64:111-26 https://doi.org/10.2307/3430003
  8. Jones JG, Lawler P, Crawley CW, Minty BD, Hulands G, Vesll N. Increased alveolar epithelial permeability in cigarette smokers. Lancet 1980;1:66-8
  9. Burns AR, Hosford SP, Dunn LA, Walker DC, Hogg JC. Respiratory epithelial permeability after cigarette exposure in guinea pigs. J Appl Physiol 1989;66:2109-16 https://doi.org/10.1152/jappl.1989.66.5.2109
  10. Lannan S, Donaldson K, Brown D, MacNee W. Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro. Am J Physiol 1994;266:L92-L100
  11. Nakano M. Free radicals and their biological significance: present and future. Hum Cell 1992;5:334-40
  12. Adams JD, Mukherjee SK, Klaidman LK, Chang ML, Yasharel R. Apoptosis and oxidative stress in the aging brain. Ann N Y Acad Sci 1996;786:135-51 https://doi.org/10.1111/j.1749-6632.1996.tb39058.x
  13. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12 https://doi.org/10.1126/science.281.5381.1309
  14. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322-6 https://doi.org/10.1126/science.281.5381.1322
  15. Leanderson P, Tagesson C. Cigarette amoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Chem Biol Interact 1992;81:197-208 https://doi.org/10.1016/0009-2797(92)90034-I
  16. Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Am J Respi Crit Care Med 1997;156:341-57 https://doi.org/10.1164/ajrccm.156.2.9611013
  17. Hall PA, Lane DP. Tumor suppressors: a developing role for p53? Curr Biol 1997;7:R144-7 https://doi.org/10.1016/S0960-9822(06)00058-3
  18. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996;10:1054-72 https://doi.org/10.1101/gad.10.9.1054
  19. MuroharaT, Kugiyama K, Ohgushi M, Sugiyama S, Yasue H. Cigarette smoke extracts contracts isolated porcine coronary arteries by superoxide anionmediated degradation of EDRF. Am J Phyiol 1994;266:H874-80
  20. Wang H, Liu X, Umino T, Skild CM, Zhu Y, Kohyama T, et al. Cigarette smoke inhibits human bronchial epithelial cell repair process. Am J Respir Cell Mol Biol 2001;25:772-9 https://doi.org/10.1165/ajrcmb.25.6.4458
  21. Xiang J, Chao DT, Korsmeyer SJ. Bax-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci U S A 1996;93:14559-63
  22. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al. Molecular characterization of mitochondrial apoptosis inducing factor. Nature 1999;397:441-6 https://doi.org/10.1038/17135
  23. Zang LY, Stone K, Pryot WA. Detection of free radicals in aqueous extracts of cigarette tar by electron spin resonance. Free Radic Biol Med 1995;19:161-7 https://doi.org/10.1016/0891-5849(94)00236-D