Radiation Oncology Journal

The Korean Society for Radiation Oncology (대한방사선종양학회)
권호 표지

Effects of Ionizing Radiation and Cisplatin on Peroxiredoxin I & II Expression and Survival Rate in Human Neuroblastoma and Rat Fibroblast Cells

전리방사선과 Cisplatin이 신경아세포종세포와 섬유모세포에서 Peroxiredoxin I과 II 발현 및 세포생존율에 미치는 영향

  • Kim, Sung-Hwan (Department of Radiation Oncology, The Catholic University of Korea) ;
  • Yoon, Sei-Chul (Department of Radiation Oncology, The Catholic University of Korea)
  • 김성환 (가톨릭대학교 의과대학 방사선종양학교실) ;
  • 윤세철 (가톨릭대학교 의과대학 방사선종양학교실)
  • Published : 2006.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

$\underline{Purpose}$: This study investigated the influence of irradiation and cisplatin on PrxI & PrxII expression and on their survival rates (SR) in SK-N-BE2C and Rat2 cell lines. $\underline{Materials\;and\;Methods}$: The amount of PrxI & PrxII production with or without N-acetyl-L-cysteine (NAC) pretreatment was studied using a western blot after 20 Gy irradiation to determine the degree of inhibition of ROS accumulation. In addition, the amount of PrxI & PrxII production after cisplatin and after combination with cisplatin and 20 Gy irradiation was studied. The SRs of the cell lines in SK-N-BE2C and Rat 2 cells, applied with 20 Gy irradiation only, with various concentrations of cisplatin and with the combination of both, were studied. The 20 Gy irradiation-only group and the combination group were each subdivided according to NAC pretreatment, and corresponding SRs were observed at 2, 6, 12 and 48 hours after treatment. $\underline{Results}$: Compared with the control group, the amount of PrxI in SK-N-BE2C increased up to 60 minutes after irradiation and slightly increased after irradiation with NAC pretreatment 60 minutes. It did not increase in Rat2 after irradiation regardless of NAC pretreatment. PrxII in SK-N-BE2C and Rat2 was not increased after irradiation regardless of NAC pretreatment. The amounts of PrxI and PrxII in SK-N-BE2C and Rat2 were not increased either with the cisplatin-only treatment or the combination treatment with cisplatin and irradiation. SRs of irradiation group with or without NAC pretreatment and the combination group with or without NAC pretreatment were compared with each other in SK-N-BE2C and Rat2. SR was significantly high for the group with increased amount of PrxI, NAC pretreatment and lower the cisplatin concentration. SR of the group in SK-N-BE2C which had irradiation with NAC pretreatment tended to be slightly higher than the group who had irradiation without NAC pretreatment. SR of the group in Rat2 which had irradiation with NAC pretreatment was significantly higher than that the group which had irradiation without NAC pretreatment. Compared to the combination group, the irradiation-only group revealed statistically significant SR decrease with the maximal difference at 12 hours. However, at 48 hours the SR of the combination group was significantly lower than the irradiation-only group. $\underline{Conclusion}$: PrxI is suggested to be an antioxidant enzyme because the amount of PrxI was increased by irradiation but decreased pretreatment NAC, a known antioxidants. Furthermore, cisplatin may inhibit PrxI production which may lead to increase cytotoxicity of irradiation. The expression of PrxI may play an important role in cytotoxicity mechanism caused by irradiation and cisplatin.

목 적: 항산화 효소인 peroxiredoxin (Prx) I과 II가 유해자극에 의해서 유발되는 세포내 반응성 산소족(reactive oxygen species, ROS)에 대한 방어기전에 관여하는지 알아보고자 이 연구를 시행하였다. 대상 및 방법: SK-N-BE2C (신경아세포종세포)와 Rat2 (섬유모세포)에서 PrxI과 PrxII 발현을 보기 위하여 방사선조사, cisplatin 단독투여 및 cisplatin-방사선조사병합투여 후에 PrxI과 PrxII에 대한 western blot을 시행하였다. 또한 N-acetyl-L-cysteine (NAC)에 의하여 PrxI과 PrxII 발현에 미치는 영향과 세포생존율을 함께 조사하였다. 두 종류 세포에 방사선조사, 다양한 농도의 cisplatin을 단독투여 및 방사선조사와 병합투여 시 생존율을 각각 분석하였고 SK-N-BE2C의 각 군에서 시간별 생존율을 관찰하였다. 결 과: PrxI의 발현은 SK-N-BE2C에서 방사선조사 후 60분까지 증가하였으나 NAC 전처치한 경우 방사선조사 후 60분에서는 대조군에 비하여 약간 증가하였다. Rat2에서는 방사선조사 후 NAC 전처치 여부에 관계없이 증가하지 않았다. PrxII의 발현은 두 가지 세포 모두에서 방사선조사와 NAC 전처치 여부에 관계없이 증가하지 않았다. SK-N-BE2C와 Rat2에서 다양한 농도의 cisplatin 단독투여나 cisplatin-방사선조사병합시에는 PrxI 및 PrxII의 발현은 증가하지 않았다. SK-N-BE2C와 Rat2에서 NAC 전처치 여부에 따른 방사선조사군 및 cisplatin-방사선조사병합군의 생존율을 각각 비교한 결과 PrxI의 발현이 증가되었고, NAC 전처치하였으며 cisplatin의 농도가 낮을수록 유의하게 생존율이 높았다. SK-N-BE2C에서 NAC를 전처치한 방사선조사군의 생존율이 NAC 전처치 안한 방사선조사군에 비하여 높은 경향만 보였으나, Rat2에서는 유의한 차이로 NAC를 전처치한 방사선조사군의 생존율이 높았다. SK-N-BE2C에서 시간별 생존율을 측정한 결과는 방사선조사군과 cisplatin-방사선조사병합군을 비교하면 방사선조사군이 빠른 세포생존율의 감소를 보였으며 12시간 때에 최대의 차이를 보였으나 48시간에서는 cisplatin-방사선조사병합군의 세포생존율이 유의하게 낮았다. 결 론: 방사선조사로 반응성 산소족이 증가되면 PrxI 발현이 증가되었으며 반응성 산소족 청소제인 NAC의 전처치에 의하여 PrxI의 발현이 감소되었다. Cisplatin은 PrxI의 발현을 억제하여 반응성 산소족에 의한 세포손상을 증가시키며 방사선으로 인한 세포치사효과를 증가시켰다고 판단된다. 이상의 결과로 PrxI의 발현여부가 방사선조사나 cisplatin의 세포치사기전에 부분적으로 관여하고 있을 것이라고 생각한다.

Keywords

References

  1. Zhang JG, Lindup WE. Differential effects of cisplatin on the production of NADH-dependent superoxide and the activity of antioxidant enzymes in rat renal cortical slices in vitro. Pharmacol Toxicol 1996;79:191-198 https://doi.org/10.1111/j.1600-0773.1996.tb02087.x
  2. Ueta E, Yoneda K, Yamamoto T, Osaki T. Manganese superoxide dismutase negatively regulates the induction of apoptosis by 5-fluorouracil, peplomycin and gamma-rays in squamous cell carcinoma cells. Jpn J Cancer Res 1999;90: 555-564 https://doi.org/10.1111/j.1349-7006.1999.tb00783.x
  3. An JH, Kim J, Seong J. Proteomics of protein expression profiling in tissues with different radiosensitivity. J Korean Soc Ther Radiol Oncol 2004;22:298-306
  4. Sun J, Chen Y, Li M, Ge Z. Role of antioxidant enzymes on ionizing radiation resistance. Free Radic Biol Med 1998;24: 586-593 https://doi.org/10.1016/S0891-5849(97)00291-8
  5. Ramu A, Cohen L, Glaubiger D. Oxygen radical detoxification enzymes in doxorubicin-sensitive and -resistant P388 murine leukemia cells. Cancer Res 1984;44:1976-1980
  6. Chlopkiewicz B, Gruber B. The in vitro study on genotoxic activity of adriamycin and bleomycin in cells of mice with different catalase and superoxide dismutase activity. Acta Pol Pharm 1997;54:437-441
  7. Chae HZ, Chung SJ, Rhee SG. Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem 1994;269:27670-27678
  8. Lee TH, Yu SL, Kim SU, Lee KK, Rhee SG, Yu DY. Characterization of mouse peroxiredoxin I genomic DNA and its expression. Gene 1999;239:243-250 https://doi.org/10.1016/S0378-1119(99)00413-8
  9. Chae HZ, Robinson K, Poole LB, Church G, Storz G, Rhee SG. Cloning and sequencing of thiol-specific antioxidant from mammalian brain: Alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc Natl Acad Sci USA 1994;91:7017-7021 https://doi.org/10.1073/pnas.91.15.7017
  10. Chae HZ, Kim HJ, Kang SW, Rhee SG. Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin. Diabetes Res Clin Pract 1999;45:101-112 https://doi.org/10.1016/S0168-8227(99)00037-6
  11. Matsumoto A, Okado A, Fujii T, et al. Cloning of the peroxiredoxin gene family in rats and characterization of the fourth member. FEBS Lett 1999;443:246-250 https://doi.org/10.1016/S0014-5793(98)01736-0
  12. Ichimiya S, Davis JG, O'Rourke DM, Katsumata M, Greene MI. Murine thioredoxin peroxidase delays neuronal apoptosis and is expressed in areas of the brain most susceptible to hypoxic and ischemic injury. DNA Cell Biol 1997;16:311-321 https://doi.org/10.1089/dna.1997.16.311
  13. Zhang P, Liu B, Kang SW, Seo MS, Rhee SG, Obeid LM. Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2. J Biol Chem 1997;272:30615-30618 https://doi.org/10.1074/jbc.272.49.30615
  14. Tuttle S, Horan AM, Koch CJ, Held K, Manevich Y, Biaglow J. Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with N-acetyl-L-cysteine or L-buthionine- S,R-sulfoximine. Int J Radiat Oncol Biol Phys 1998;42:833-888 https://doi.org/10.1016/S0360-3016(98)00331-9
  15. Seo MS, Kim JK, Lim Y, et al. Rapid degradation of PrxI and PrxII induced by silica in Rat2 cells. Biochem Biophys res Commun 1999;265:541-544 https://doi.org/10.1006/bbrc.1999.1709
  16. Yamaoka K, Kojima S, Nomura T. Changes of SOD-like substances in mouse organs after low-dose X-ray irradiation. Physiol Chem Phys Med NMR 1999;31:23-28
  17. Gralla JD, Sasse-Dwight S, Poljak LG. Formation of blocking lesions at identical DNA sequences by the nitrosourea and platinum classes of anticancer drugs. Cancer Res 1987;47:5092-5096
  18. Rosenberg J, Sato P. Messenger RNA loses the ability to direct in vitro peptide synthesis following incubation with cisplatin. Mol Pharmacol 1988;33:611-616
  19. Corda Y, Anin MF, Leng M, Job D. RNA polymerases react differently at d (ApG) and d (GpG) adducts in DNA modified by cis-diamminedichloroplatinum (II). Biochemistry 1992;31:1904-1908 https://doi.org/10.1021/bi00122a002
  20. Caney C, Bulmer JT, Singh G, Lukka H, Rainbow AJ. Pre-exposure of human squamous carcinoma cells to low- doses of gamma-rays leads to an increased resistance to subsequent low-dose cisplatin treatment. Int J Radiat Biol 1999;75:963-972 https://doi.org/10.1080/095530099139728
  21. Dempke WC, Shellard SA, Hosking LK, Fichtinger- Schepman AM, Hill BT. Mechanisms associated with the expression of cisplatin resistance in a human ovarian tumor cell line following exposure to fractionated X-irradiation in vitro. Carcinogenesis 1992;13:1209-1215 https://doi.org/10.1093/carcin/13.7.1209
  22. Cregan SP, Smith BP, Brown DL, Mitchel RE. Two pathways for the induction of apoptosis in human lymphocytes. Int J Radiat Biol 1999;75:1069-1086 https://doi.org/10.1080/095530099139539
  23. Giusti AM, Raimondi M, Ravagnan G, Sapora O, Parasassi T. Human cell membrane oxidative damage induced by single and fractionated doses of ionizing radiation: a fluorescence spectroscopy study. Int J Radiat Biol 1998; 74:595-605 https://doi.org/10.1080/095530098141177