Journal of Oral Medicine and Pain

Korean Academy of Orofacial Pain and Oral Medicine (대한안면통증구강내과학회)
권호 표지

Expression of Bcl-2 Family in 4-Nitroquinoline 1-Oxide-Induced Tongue Carcinogenesis of the Rat

백서 혀에서의 4-nitroquinoline 1-oxide 유도 발암과정에서 Bcl-2 계 유전자의 발현

  • Choi, Jae-Wook (Dept. of Oral Medicine, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Chung, Sung-Su (Dept. of Anesthesiology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Lee, Geum-Sug (Dept. of Oral Medicine, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Kim, Byung-Gook (Dept. of Oral Medicine, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Kim, Jae-Hyeong (Dept. of Oral Medicine, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Kook, Eun-Byul (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Jang, Mi-Sun (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Ko, Mi-Kyeong (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Jung, Kwon (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Choi, Hong-Ran (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University) ;
  • Kim, Ok-Joon (Dept. of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University)
  • 최재욱 (전남대학교 치의학전문대학원 구강내과학교실, 전남대학교 치의학연구소) ;
  • 정성수 (전남대학교 치의학전문대학원 마취통증의학교실, 전남대학교 치의학연구소) ;
  • 이금숙 (전남대학교 치의학전문대학원 구강내과학교실, 전남대학교 치의학연구소) ;
  • 김병국 (전남대학교 치의학전문대학원 구강내과학교실, 전남대학교 치의학연구소) ;
  • 김재형 (전남대학교 치의학전문대학원 구강내과학교실, 전남대학교 치의학연구소) ;
  • 국은별 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소) ;
  • 장미선 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소) ;
  • 고미경 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소) ;
  • 정권 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소) ;
  • 최홍란 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소) ;
  • 김옥준 (전남대학교 치의학전문대학원 구강병리학교실, 전남대학교 치의학연구소)
  • Published : 2005.09.30
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Abstract

The number of patients with tongue carcinoma is increasing rapidly among young individuals in many parts of the world. Oral carcinoma progresses from hyperplastic lesion through dysplasia to invasive carcinoma and the concept of "field cancerization" with molecular alteration has been suggested for oral cavity carcinogenesis. Significant improvement in treatment and prognosis will depend on more detailed understanding of the multi-step process leading to cancer development. To induce tongue carcinoma in rat by 4-NQO, each drinking water was made to 10 ppm, 25 ppm, 50 ppm and control (only D.W. without 4-NQO). Specimens were classified into 4 groups such as control, I (mild & moderate dysplasia), II (severe dysplasia and carcinoma in situ), III (carcinoma). The mRNA expressions of Bcl-2 family were evaluated by RT-PCR technique. For anti-apoptotic Bcl-2 family, mRNA expression of Bcl-w was down-regulated in all stages of tongue carcinogenesis model. However, mRNA expression of Bcl-2 was up-regulated. For pro-apoptotic Bcl-2 family, all members were down-regulated in all stages of tongue carcinogenesis model except for Bad mRNA in group III. In terms of BH3 only protein, mRNA expressions of Bok and Mcl-1 were down regulated in all stages of specimen, but Bmf in group II and BBC3 in group III were up-regulated. Our current findings demonstrated the involvements of mRNA expression of Bcl-2 family in multi-step tongue carcinogensis. This highlights the necessity for continued efforts to discover suitable biomakers (Bcl-2 family) for early diagnosis of the disease, and to understand its pathogenesis as a first step in improving methods of treatment. The discovery of these potential biomarkers and molecular targets for cancer diagnostics and therapeutics has the potential to significantly change the clinical approach and outcome of the disease.

전 세계적으로 구강암의 빈도는 점점 증가 추세이며, 특히 한국인의 있어 혀(tongue)는 구강암이 가장 호발하는 장소이다. 구강암은 발암 단계에서부터 과증식 병소(hyperplastic lesion), 이형성(dysplasia) 및 상피내암(carcinoma in situ) 을 거쳐 악성 암종으로 발전하는 다단계 발암과정을 보이며, 분자 생물학적 변이가 구강암을 진행시킴이 널리 알려져 있다. 또한, 구강암은 일반적으로 암세포의 증식 및 고사(apoptosis)의 억제가 중요한 역할을 하고 있다 알려져 있다. 그리고, Bcl-2 family 는 세포 고사에 주요한 역할을 하고 있음이 알려져 있다. 그러나, 이들과 관련한 구강암 발생과정의 변화에 대해서는 널리 연구된 바가 없다. 본 연구는 백서에서 발암 물질인 4-NQO로 구강암을 유도시키고, 구강암 발생 다단계별로 Bcl-2 family의 mRNA 변화를 RT-PCR을 이용해 살펴보았다. Bcl-2 family는 크게 3군, 즉 1) anti-apoptotic, 2) pro-apoptotic, 그리고 3) BH3 only protein으로 분류할 수 있으며, 본 연구에서 anti-apoptotic molecules인 Bcl-w는 모든 군에서 발현이 감소되었으며, Bcl-2는 발현이 증가 되었다. pro-apoptotic molecules에서는 Bad가 제 3군 (편평세포암종)에서 발현이 증가 되었고, 나머지는 감소하였다. BH-3 only protein에서는 Bmf가 제 2군에서, BBC3가 제 3군에서 발현이 증가하였고, 나머지는 모든 군에서 감소하였다. 결론적으로, 4-NQO로 유도된 백서의 발암단계에서, Bcl-2 family의 mRNA 양상은 다양하게 관찰되었으나, Bad 및 BBC3 mRNA가 제 3군에서, Bmf mRNA가 제 2군에서의 발현이 특별함을 알 수 있어, 다단계 발암과정에서의 구강암을 진단하는데 유용하리라 사료된다.

Keywords

References

  1. Calfano, J., van der Riet, P., Westra, W., Nawroz, H., Clayman,G., Piantadosi, S., Corio, R., Lee, D., Greenberg, B., Koch, W. and Sidransky, D. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res. 1996;56: 2488-2492
  2. Slaughter, D.L., Southwick, H.W., Smejkal, W. Filed cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer. 1953;6: 963-968 https://doi.org/10.1002/1097-0142(195309)6:5<963::AID-CNCR2820060515>3.0.CO;2-Q
  3. Patridge, M., Emilion, G., Pateromichelakis, S., Phillips, E., and Langdon, J. Field cancerisation of the oral cavity: comparison of the spectrum of molecular alrerations in cases presention with both dysplastic and malignant lesions. Oral. Oncel. 1997;33: 332-337 https://doi.org/10.1016/S1368-8375(97)00035-3
  4. Shklar, G. Development of experimental oral carcinogenesis and its impact on current oral cancer research. J. Dent. Res. 1999;78: 1768-1772 https://doi.org/10.1177/00220345990780120101
  5. Eveson, J.W. Animal models of intra-oral chemical carcinogenesis: a review. J. Oral. Pathol. 1981;10: 129-146 https://doi.org/10.1111/j.1600-0714.1981.tb01259.x
  6. Fisker, A.V. Experimental oral carcinogenesis. A basic rat model for the study of oral carcinogenesis using the carcinogen 4-nitroquinoline 1-oxide. Dan. Med. Bull. 1990;15:43-47
  7. Steidler, N.E., and Reade, P.C. Initiation and promotion of experimental oral mucosal carcinogenesis in mice. J. Oral. Pathol. 1986;15: 43-47 https://doi.org/10.1111/j.1600-0714.1986.tb00562.x
  8. Tanaka, T., Kawamori, T., Ohnishi, M., Okamoto, K., Mori, H. and Hara, A. Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis by dietary protocatechuic acid during initiation and postinitiation phases. Cancer Res. 1994;54: 2359-2365
  9. Ohne, M., Satoh, T., Yamada, S. and Takai, H. Experimental tongue carcinoma of rats induced by oral administration of 4-nitroquinoline 1-oxide (4-NQO) in drinking water. Oral Surg. Oral Med. Oral Pathol. 1985;59: 600-607 https://doi.org/10.1016/0030-4220(85)90189-6
  10. Wallenius, K. and Lekholm, U. Oral cancer in rats induced by the water soluble carcinogen 4-nitroquinoline 1-oxide. Odont. Rev. 1973;24: 39-48
  11. Moore, M.A. and Tsuda, H. Chronically elevated proliferation as a risk factor for neoplasia. Eur. J. Cancer Prev. 1998;7: 353-385 https://doi.org/10.1097/00008469-199810000-00002
  12. Mori, H., Sugie, S., Yoshimi, N., Hara, A. and Tanaka, T. control of cell proliferation in cancer prevention. Mutat Res. 1999;428: 291-298 https://doi.org/10.1016/S1383-5742(99)00055-1
  13. Sharma, R.A., Manson, M.M., Gescher, A. and Steward, W.P. Colorectal cancer chemoprevention: biochemical targets and clinical development of promising agents. Eur. J. Cancer. 2001;37: 12-22
  14. Adams, J.M., Cory, S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281: 1322-1326 https://doi.org/10.1126/science.281.5381.1322
  15. Wei, M.C., Zong, W.X., Cheng, E.H., Lindsten, T., Panoutsakopoulou, V., Ross, A.J., Roth, K.A., Macgregor, G.R., Thompson, C.B., and Korsmeyer, S.J. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 1998;292: 727-730
  16. Zong, W.X.,, Lindsten, T., Ross, A.J., MacGregor. G.R., Thompson, C.B. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev. 2001;15: 1481-1486 https://doi.org/10.1101/gad.897601
  17. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., Martinou, J.C. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J. 2000 ;345 Pt 2: 271-278 https://doi.org/10.1042/0264-6021:3450271
  18. Jurgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., Reed, J.C. Bax directly induces release of cytochrome c from isolated mitochondria.Proc Natl Acad Sci U S A. 1998;95: 4997-5002
  19. Wei, M.C., Lindsten, T., Mootha, V.K., et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 2000;14: 2060-71
  20. Reed, J.C. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994;124: 1-6 https://doi.org/10.1083/jcb.124.1.1
  21. Reed, J.C., Miyashita, T., Takayama, S., et al. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy.J Cell Biol. 1996;60: 23-32
  22. Ziwei, H. Bcl-2 proteins as target for anticancer durg design. Oncogene. 2000;19: 6627-6631 https://doi.org/10.1038/sj.onc.1204087
  23. Mikhailov, V., Mikhailova, M., Pulkrabek, D.J., Dong, Z., Venkatachalam, M.A., Saikumar, P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem. 2001;276:18361-18374 https://doi.org/10.1074/jbc.M100655200
  24. Murphy, K.M., Streips, U.N., Lock, R.B. Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation. J Biol Chem. 2000;275:17225-17228 https://doi.org/10.1074/jbc.C900590199
  25. Akao, Y., Otsuki, Y., Kataoka, S., Ito, Y., Tsujimoto, Y. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 1994;54: 2468-2471
  26. Foyouzi-Youssefi, R., Arnaudeau, S., Borner, C., et al. Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2000;97: 5723-5728
  27. He, H., Lam, M., McCormick, T.S., Distelhorst, C.W. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol. 1997;138: 1219-28 https://doi.org/10.1083/jcb.138.6.1219
  28. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R.L., Distelhorst, C.W.. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci U S A. 1994;91: 6569-6573
  29. Nutt, L.K., Pataer, A., Pahler, J., et al. Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores. J Biol Chem. 2002;277: 9219-9225 https://doi.org/10.1074/jbc.M106817200
  30. Pinton, P., Ferrari, D., Magalhaes, P., et al. Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells. J Cell Biol. 2000;148: 857-862 https://doi.org/10.1083/jcb.148.5.857
  31. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 ;74: 609-19 https://doi.org/10.1016/0092-8674(93)90509-O
  32. Oltvai, Z.N., Korsmeyer, S.J. Checkpoints of dueling dimers foil death wishes. Cell. 1994 ;79: 189-92 https://doi.org/10.1016/0092-8674(94)90188-0
  33. John, CR. Bcl-2 family proteins. Oncogene. 1998;17: 3225-3236 https://doi.org/10.1038/sj.onc.1202591
  34. Hsu, S.Y., Hsueh, A.J. A splicing variant of the Bcl-2 member Bok with a truncated BH3 domain induces apoptosis but does not dimerize with antiapoptotic Bcl-2 proteins in vitro. J Biol Chem. 1998;273: 30139-30146 https://doi.org/10.1074/jbc.273.46.30139
  35. Show, M.D., Folmer, J.S., Anway, M.D., Zirkin, B.R. Testicular expression and distribution of the rat bcl2 modifying factor in response to reduced intratesticular testosterone. Biol Reprod. 2004;70: 1153-1161 https://doi.org/10.1095/biolreprod.103.023200
  36. Yu, J., Zhang, L., Hwang, P.M., Kinzler, K.W., Vogelstein, B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol. Cell. 2001;7: 673-682 https://doi.org/10.1016/S1097-2765(01)00213-1
  37. Nakano, K., Vousden,K.H. PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell. 2001; 7: 683-694 https://doi.org/10.1016/S1097-2765(01)00214-3
  38. Reed JC. Mechanisms of apoptosis avoidance in cacner. Curr Opin Oncol 1999; 11:68-75 https://doi.org/10.1097/00001622-199901000-00014
  39. Okazaki Y, Tanaka Y, Tonogi M, Yamane G. Ivestigation of environmental factors for diagnosting malignant potential in oral epithelial dysplasia
  40. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68: 251-306 https://doi.org/10.1016/S0074-7696(08)62312-8
  41. Ravi D, Nalinakumari KR, Rajaram RS, Nair MK, Pillai MR. Expression of programmed cell death regulatory p53 and bcl-2 proteins in oral lesions. Cancer Lett. 1996;105(2):139-46 https://doi.org/10.1016/0304-3835(96)04258-9
  42. Jordan RC, Catzavelos GC, Barrett AW, Speight PM. Differential expression of bcl-2 and bax in squamous cell carcinomas of the oral cavity. Eur J Cancer B Oral Oncol. 1996;32(6):394-400 https://doi.org/10.1016/S0964-1955(96)00033-4
  43. Koide N, Koike S, Adachi W, Amano J, Usuda N, Nagata T. Immunohistochemical expression of bcl-2 protein in squamous cell carcinoma and basaloid carcinoma of the esophagus. Surg Today. 1997;27(8):685-91 https://doi.org/10.1007/BF02384977
  44. Nakopoulou L, Vourlakou C, Zervas A, Tzonou A, Gakiopoulou H, Dimopoulos MA. The prevalence of bcl-2, p53, and Ki-67 immunoreactivity in transitional cell bladder carcinomas and their clinicopathologic correlates. Hum Pathol. 1998;29(2):146-54 https://doi.org/10.1016/S0046-8177(98)90225-8
  45. Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, Reed JC. Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am J Pathol. 1994 ;145(6):1323-36
  46. Chrysomali E, Greenspan JS, Dekker N, Greenspan D, Regezi JA. Apoptosis-associated proteins in oral hairy leukoplakia. Oral Dis. 1996 ;2(4):279-84 https://doi.org/10.1111/j.1601-0825.1996.tb00238.x
  47. Loro LL, Vintermyr OK, Liavaag PG, Jonsson R, Johnnessen AC. Oral squamous cell carcinoma is associated with decreased bcl-2/bax expression ratio and incresed apoptosis. Hum Pathol ;30:1097-1105 https://doi.org/10.1016/S0046-8177(99)90229-0
  48. O'Reilly L. A., Print C., Hausmann G., et al. Tissue expression and subcellular localization of the pro-survival molecule Bcl-w. Cell Death Differ., 2001;8:486-494 https://doi.org/10.1038/sj.cdd.4400835
  49. Wilson JW, Nostro MC, Balzi M, et al. Bcl-w expression in colorectal adenocarcinoma. Br J Cancer. 2000;82(1):178-85 https://doi.org/10.1054/bjoc.1999.0897
  50. Lee HW, Lee SS, Lee SJ, Um HD. Bcl-w is expressed in a majority of infiltrative gastric adenocarcinomas and suppresses the cancer cell death by blocking stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Cancer Res. 2003;63(5):1093-100
  51. Li D, Ueta E, Kimura T, Yamamoto T, Osaki T. Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination. Cancer Sci. 2004;95(8):644-50 https://doi.org/10.1111/j.1349-7006.2004.tb03323.x
  52. Masuda M, Suzui M, Lim JT, Weinstein IB Epigallocatechin-3-gallate inhibits activation of HER-2/neu and downstream signaling pathways in human head and neck and breast carcinoma cells. Clin Cancer Res. 2003;9(9):3486-91
  53. Itoh M, Noutomi T, Chiba H, Mizuguchi J. BcI-xL antisense treatment sensitizes Bcl-xL-overexpressing squamous cell carcinoma cells to carboplatin. Oral Oncol. 2002 ;38(8):752-6 https://doi.org/10.1016/S1368-8375(02)00047-7
  54. Mihara M, Shintani S, Kiyota A, Matsumura T, Wong DT. Cyclin-dependent kinase inhibitor (roscovitine) suppresses growth and induces apoptosis by regulating Bcl-x in head and neck squamous cell carcinoma cells. Int J Oncol. 2002 ;21(1):95-101
  55. M.C. Kiefer, M.J. Brauer, V.C. Powers et al., Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature 2002 ;374: 736 - 739 https://doi.org/10.1038/374736a0
  56. S. Krajewski, M. Krajewska and J.C. Reed, Immunohistochemical analysis of in vivo patterns of Bak expression, a proapoptotic member of the Bcl-2 protein family. Cancer Research 1996; 56(12): 2849-2855
  57. M. Krajewska, S.F. Moss, S. Krajewski, K Song, P.R. Holt and J.C. Reed, Elevated expression of Bcl-X and reduced Bak in primary colorectal adenocarcinomas. Cancer Research 1996; 56(10) : 2422–2427
  58. M. Krajewska, C.M. Fenoglio-Preiser, S. Krajewski et al., Immunohistochemical analysis of Bcl-2 family proteins in adenocarcinomas of the stomach. American Journal of Pathology 1996; 149(5): 1449-1457
  59. Xie X, Clausen OP, Boysen M. Prognostic value of Bak expression in oral tongue squamous cell carcinomas. Oncol Rep. 2003 ;10(2):369-74
  60. Huang DC, Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell. 2000;103(6):839-42 https://doi.org/10.1016/S0092-8674(00)00187-2
  61. Ferrer A, Marce S, Bellosillo B, et al. Activation of mitochondrial apoptotic pathway in mantle cell lymphoma: high sensitivity to mitoxantrone in cases with functional DNA-damage response genes. Oncogene. 2004; 23(55) : 8941-9. https://doi.org/10.1038/sj.onc.1208084
  62. Nakano K, Vousden KH. PUMA, a novel porapoptotic gene, is induced by p53. Molecular Cell 2001;7: 683-694 https://doi.org/10.1016/S1097-2765(01)00214-3
  63. Han J, Flemington C, Houghton AB, et al. Expression of bbc3, a pro-apoptotoic Bch3-only gene, is regulated by diverse cell death and survival signals. Proceedings of the National Academy of Sciences of the USA. 2001;98:11318-11323