Journal of the Korean Association of Oral and Maxillofacial Surgeons

  • 제29권5호
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  • Pages.272-281
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  • 2003
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  • 2234-7550(pISSN)
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  • 2234-5930(eISSN)
대한구강악안면외과학회 (The Korean Association of Oral and Maxillofacial Surgeons)
권호 표지

Curcumin과 resveratrol에 의한 두경부암 유래의 HN-4 세포의 세포주기, 세포사 및 전이관련 단백질의 발현 조절

EFFECT OF CURCUMIN AND RESVERATROL ON THE CELL CYCLE REGULATION, APOPTOSIS AND INHIBITION OF METASTASIS RELATED PROTEINS IN HN-4 CELLS

  • 김사엽 (경북대학교 치과대학 구강악안면외과학교실) ;
  • 이상한 (경북대학교 치과대학 구강악안면외과학교실) ;
  • 권택규 (계명대학교 의과대학 면역학교실)
  • Kim, Sa-Yub (Dept. of Oral & Maxillofacial Surgery, School of Dentistry, Kyungpook National University) ;
  • Lee, Sang-Han (Dept. of Oral & Maxillofacial Surgery, School of Dentistry, Kyungpook National University) ;
  • Kwon, Taeg-Kyu (Dept. of Immunology, School of Medicine, Kyemyung University)
  • 발행 : 2003.10.30
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초록

본 연구에서는 두경부암 HN-4 세포에서 cancer chemopreventive agent인 curcumin과 resveratrol를 처리하여 HN-4 세포의 생육 억제의 원인이 apoptotic cell death에 의하여 일어나며 세포 주기 조절 단백질의 발현 및 암 전이에 관련된 MMP 활성 저해 기전에 대하여 이해하고자 하였다. HN-4 세포에 다양한 농도(10-100 ${\mu}M$) curcumin을 처리하여 세포주기 조절 단백질의 발현을 측정한 결과 cdk1과 cdk4 단백질이 농도 의존적으로 발현이 감소하였으며, resveratrol 처리에서는 cyclin A 단백질의 특이적인 감소 현상을 확인하였다. Curcumin에 의한 apoptosis 유도 기전을 조사한 결과 anti-apoptotic 기능이 있는 Bcl-2 및 Bcl-xL 단백질 발현 감소 현상은 없었으나, caspase 저해 IAP family 단백질중 cIAP1과 survivin 단백질 발현 현상이 처리 농도 의존적으로 감소하였다. Resveratrol을 처리한 경우 Bcl-2 및 survivin 단백질 발현 감소현상을 확인하였다. Curcumin과 resveratrol에 의한 apoptosis 과정은 caspase-3 의존적인 apoptosis 유도 기전을 보였다. Curcumin에 의한 apoptosis 과정은 항산화제인 NAC 처리에 의해서 저해되었다. Curcumin과 NAC 동시 처리는 cytochrome c 유리, caspase-3 활성화 및 Bax 단백질 분절 현상을 억제하였다. 그러나 resveratrol에 의한 apoptosis 과정은 NAC 처리에 의하여 억제되지 않았다. Curcumin과 resveratrol에 의한 암 전이 관련 단백질인 MMP2와 9의 활성 저해효과도 확인하였다. 결론적으로, curcumin에 의한 항암효과는 세포주기 조절 및 apoptosis 유도 및 전이 관련 단백질의 활성 억제를 통하여 야기되는 것으로 생각되어 진다.

Nontraditional or alternative medicine is becoming an increasingly attractive approach for the treatment of various inflammatory disorders and cancers. Curcumin is the major constitute of turmoric powder extracted from the rhizomes of the plant Curcuma longa. Resveratrol is a phytoalexin present in grapes and a variety of medicinal plants. In this report, We investigated the effect of curcumin and resveratrol on regulatory protein of cell cycle, induction of apoptosis and MMP activity. Treatment with 75 M curcumin for 24 hrs produced morphological changing in HN-4 cells. Curcumin and resveratrol inhibited the cellular growth in HN-4 cells. Inhibition of cell growth was associated with down-regulation of cell cycle regulatory proteins. Curcumin-induced caspase-3 activation and Bax degradation were dose-dependent with a maximal effect at a concentration of 100 M. The elevated caspase-3 activity in curcumin treated HN-4 cells are correlated with down-regulation of survivin and cIAP1, but not cIAP2. Curcumin induced a dose-dependent increase of cytochrome c in the cytosol. Curcumin induced-apoptosis was mediated through the release of cytochrome c. In addition, curcumin-induced apoptosis was caused by the generation of reactive oxygen species, which was prevented by antioxidant N-acetyl-cysteine (NAC). Cotreatment with NAC markedly prevented cytochrome c release, Bax cleavage and cell death. Also resveratrol-induced apoptosis was preceded by down-regulation of the anti-apoptotic Bcl-2, cIAP1, and caspase-3 activity. However, resveratrol-induced apoptosis was not prevented by antioxidant NAC. In addition, HN-4 cells release basal levels of MMP2 when cultured in serum-free medium. Treatment of the cells with various concentrations of PMA for 24 hr induced the expression and secretion of latent MMP9 as determined by gelatin zymography. HN-4 cells were treated with various concentrations of curcumin and resveratrol in the presence of 75 nM PMA, and MMP2 and 9 activities were inhibited by curcumin and resveratrol. These findings have implications for developing curcumin-based anticancer and anti-inflammation therapies.

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참고문헌

  1. Kelloff GJ, Johnson JR, Crowell JA, Boone CW, DeGeorge JJ, Steele VE, Mehta MU, Temeck JW, Schmidt WJ, Burke G, and Sigman CC : Approaches to the development and marketing approval of drugs that prevent cancer. Cancer Epidemiol. Biomarkers Prev., 1995 ; 4 :1-10.
  2. Ames BN, Gold LS, and Willett WC : The causes and prevention of cancer. Proc. Natl. Acad. Sci. U. S. A., 1995 ; 92 : 5258-5265.
  3. Ammon HP, and Wahl MA : Pharmacology of Curcuma longa. Planta. Med., 1991 ; 57 :1-7.
  4. Huang MT, Smart RC, Wong CQ, and Conney AH : Inhibitory effect of curcumin, chlorogenic acid, caffeicacid, and ferulic acid on tumor promotion in mouse skin by\12-O-tetradecanoylphor bol-13-acetate. Cancer Res., 1998 ; 48 : 5941-5946.
  5. Huang MT, Newmark HL, and Frenkel K : Inhibitory effects of curcumin on tumorigenesis in mice. J. Cell Biochem. Suppl., 1997 ; 27 : 26-34.
  6. Soleas, GJ., Diamandis, EP., and Goldberg, DM. : Wine as a biological fluid:history, production, and role in disease prevention. J. Clin. Lab. Anal., 1997 ; 11: 287-313.
  7. Soleas, GJ., Diamandis, EP., and Goldberg, DM. : Resveratrol: a molecule whose time has come? And gone? Clin. Biochem., 1997 ; 30 : 91-113.
  8. Fremont, L. : Biochemical effect of resveratrol. Life Sci., 2000 ; 66 : 6663-6673.
  9. Jang, M., Cai, L., Udeani, GO., Slowing, KV., Thomas, CF., Beecher, CW. Fong, HH., Farnsworth, NR., Kinghorn, AD., Mehta, RG., Moon, RC., and Pezzuto, JM. : Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 1997 ; 275 : 218-220. https://doi.org/10.1126/science.275.5297.218
  10. Fauconneau, B., Waffo-Teguo, P., Huguet, F. Barrier, L., Decendit, A., and Merillon, JM. : Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci., 1997 ; 61 : 2103-2110.
  11. Pace-Asciak, CR., Rounova, O., Hahn, SE., Diamandis, EP., and Goldberg, DM. : The red wine phenolics trans resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis :Implication for protection against coronary heart disease. Clin. Chim. Acta., 1996 ; 246 :163-182.
  12. Mgbonyebi, OP., Russo, J., and Russo, IH. : Antiproliferative effect of synthetic resveratrol on human breast epithelial cells. Int. J. Oncol., 1998 ; 12 : 865-869.
  13. Clement, MV., Hirpara, JL., Vhawdhury, SH., and Pervaiz, S. : Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood, 1998 ; 92: 996-1002.
  14. Surh, YJ., Hurh, YJ., Kang, JY., Lee, E., Kong, G., and Lee, SJ. : Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL60) cells. Cancer letters, 1999 ; 140:1-10. https://doi.org/10.1016/S0304-3835(99)00039-7
  15. Ragione, FD., Cucciolla, V., Borriello, A., Pietra, VD., Racioppi, L., Soldati, G., Manna, C., Galletti, P., and Zappia, V. : Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochem. Biophys. Res. Commun., 1998 ; 250: 53-58.
  16. Hunter, T. and Pines, J. : Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell, 1994 ; 79: 573-82.
  17. Morgan, DO. : Principles of CDK regulation. Nature, 1995 ; 374: 131-134. https://doi.org/10.1038/374131a0
  18. Sherr, CJ. and Roberts, JM. : Inhibitors of mammalian G1 cyclindependent kinases. Genes Dev. 1995 ; 9: 1149-1163.
  19. Ikeda, MA., Jakoi, L. and Nevins, JR. : A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation. Proc. Natl. Acad. Sci. U. S. A. 1996 ; 93: 3215-3220.
  20. Moberg, K., Starz, MA. and Lees, JA. : E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry. Mol. Cell. Biol. 1996 ; 16: 1436-1449.
  21. Wyllie, AH. : Apoptosis. Br. J. Cancer, 1993 ; 67: 205-208.
  22. Thompson, CB. : Apoptosis in the pathogenesis and treatment of disease. Science, 1995 ; 267: 1456-1462. https://doi.org/10.1126/science.7878464
  23. Tsujimoto, Y., Jaffe, E., Cossman, J., Gorham, J., Nowell, PC. and Croce, CM. : Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation. Nature, 1985 ; 315: 340-343.
  24. Tsujimoto, Y. and Croce, CM. : Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. U. S. A. 1986 ; 83: 5214-5218.
  25. Lawrence, MS., Ho, DY., Sun, GH.,Steinberg, GK. and Sapolsky, RM. : Over expression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo. J. Neurosci., 1996 ; 16: 486-496.
  26. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H. and Tsujimoto, Y. : Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature, 1995 ; 374: 811-813.
  27. Tudor, G., Aguilera, A,. Halverson, DO. ,Laing, ND. and Sausville, EA. : Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels. Cell Death Differ., 2000 ; 7: 574-586.
  28. illardon, F., Moll, I., Meyer, M. and Michaelidis, TM. : Alterations in cell death and cell cycle progression in the UV-irradiated epidermis of bcl-2-deficient mice. Cell Death Differ., 1999 ; 6: 55-60.
  29. Kyprianou, N., King, ED., Bradbury, D. and Rhee, JG. : bcl-2 overexpression delays radiation-induced apoptosis without affecting the clonogenic survival of human prostate cancer cells. Int. J. Cancer, 1997 ; 70: 341-348.
  30. Yang, J., Liu, X., Bhalla, K., Kim, CN., Ibrado, AM., Cai, J., Peng, TI., Jones, DP. and Wang, X. : Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science, 1997 ; 275: 1129-1132.
  31. Kluck, RM., Bossy-Wetzel, E., Green, DR. and Newmeyer, DD. : The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science, 1997 ; 275: 1132-1136.
  32. Koriyama, H., Kouchi, Z., Umeda, T., Saido, TC., Momoi, T., Ishiura, S. and Suzuki, K. : Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis. Cell Signal., 1999 ; 11: 831-838.
  33. Park, JW., Choi, YJ., Suh, SI., Baek, WK., Suh, MH., Jin, IN., Min, DS., Woo, JH., Chang, JS., Passaniti, A., Lee, YH. and Kwon, TK. : Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis, 2001 ; 22: 1633-1639.
  34. Mashima, T., Naito, M., Noguchi, K., Miller, DK., Nicholson, DW. and Tsuruo, T.: Actin cleavage byCPP-32/apopain during the development of apoptosis. Oncogene, 1997 ; 14: 1007-1012.
  35. Decker, P., Isenberg, D. and Muller, S. : Inhibition of caspase-3-mediated poly(ADP-ribose) polymerase (PARP) apoptotic cleavage by human PARP autoantibodies and effect on cells undergoing apoptosis. J. Biol. Chem., 2000 ; 275: 9043-9046.
  36. An, B. and Dou, QP. : Cleavage of retinoblastoma protein during apoptosis: an interleukin 1 beta-converting enzyme-like protease as candidate. Cancer Res., 1996 ; 56: 438-442.
  37. Zhang, Y., Fujita, N. and Tsuruo, T. : Caspase-mediated cleavage of p21Waf1/Cip1 converts cancer cells from growth arrest to undergoing apoptosis. Oncogene, 1999 ; 18: 1131-1138.
  38. John, A. and Tuszynski, G. : The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol. Oncol. Res., 2001 ; 7: 14-23.
  39. Stamenkovic, I. : Matrix metalloproteinases in tumor invasion and metastasis. Semin. Cancer Biol., 2000 ; 10: 415-433.
  40. Curran, S. and Murray, G. I. : Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis. Eur. J. Cancer, 2000 ; 36: 1621-1630.
  41. Nelson, AR., Fingleton, B., Rothenberg, ML. and Matrisian, LM. : Matrix metalloproteinases: biologic activity and clinical implications. J. Clin. Oncol., 2000 ; 18: 1135-4119.
  42. Park, JW., Choi, YJ., Jang, MA., Lee, YS., Jun, DY., Suh, SI., Baek, WK., Suh, MH., Jin, IN. and Kwon, TK. : Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells. Cancer Lett., 2001 ; 163: 43-49.
  43. Chung, LY., Cheung, TC., Kong, SK., Fung, KP., Choy, YM., Chan, ZY. and Kwok, TT. : Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells. Life Sci., 2001 ; 68: 1207-1214.
  44. Islam, S., Islam, N., Kermode, T., Johnstone, B., Mukhtar, H., Moskowitz, RW., Goldberg, VM., Malemud, CJ. and Haqqi, TM. : Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells Biochem. Biophys. Res. Commun., 2000 ; 270:793-797.
  45. Grutter, MG. : Caspases: key players in programmed cell death. Curr. Opin. Struct. Biol., 2000 ; 10: 649-655.
  46. Nunez, G., Benedict, MA., Hu, Y. and Inohara. N. : Caspases: the proteases of the apoptotic pathway. Oncogene, 1998 ; 17: 3237-3245.
  47. Srinivasula, SM., Ahmad, M., Fernandes-Alnemri, T. and Alnemri, ES. :Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell, 1998 ; 1: 949-957.
  48. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, SM., Ahmad, M., Alnemri, ES. and Wang, X. : Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 1997 ; 91: 479-489.