Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

The Effects of Fucoidan on the Activation of Macrophage and Anticancer in Gastric Cancer Cell

Fucoidan의 면역세포 활성 및 위암 세포주에서의 항암효과

  • An, In-Jung (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Cho, Sung-Dae (Department of Oral Pathology, School of Dentistry, Institute of Oral Bioscience, Chonbuk National University) ;
  • Kwon, Jung-Ki (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Hye-Ri (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Yu, Hyun-Ju (Department of Oral Pathology, School of Dentistry, Institute of Oral Bioscience, Chonbuk National University) ;
  • Jung, Ji-Youn (Department of Companion and Laboratory Animal Science, Kongju National University)
  • 안인정 (공주대학교 특수동물학과) ;
  • 조성대 (전북대학교 구강병리학교실) ;
  • 권중기 (공주대학교 특수동물학과) ;
  • 김혜리 (공주대학교 특수동물학과) ;
  • 유현주 (전북대학교 구강병리학교실) ;
  • 정지윤 (공주대학교 특수동물학과)
  • Received : 2012.09.26
  • Accepted : 2012.11.09
  • Published : 2012.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

This study was designed to investigate the effect of fucoidan on the activation of macrophage and on induction of apoptosis in AGS cell. To measure the activity of macrophages, NO and TNF-${\alpha}$ assays were performed in Raw 264.7 cell. Treatment with fucoidan significantly increased production of NO and TNF-${\alpha}$, indicating activation of macrophages. The result of MTT assay shows that cell viability was significantly decreased in a dose and time-dependent manner. Fucoidan increased to enhance mitochondrial membrane permeability, as well as the cytochrome c release from the mitochondria. Fucoidan decreased Bcl-2 and XIAP expression, whereas the expression of Bax was increased in a time-dependent manner compared to the control. In addition, the active forms of caspase-9 were increased, and the inactivation of Akt was decreased in a time-dependent manner. Caspase inhibitor, z-VAD-FMK, canceled the apoptosis of fucoidan, expression of Bax and caspase-9 were decrease. These results indicate that fucoidan induces activation of macrophage and apoptosis through activation of caspase on AGS cell.

본 연구에서는 fucoidan이 macrophage의 활성과 항암효과를 확인하기 위하여 수행되었다. Fucoidan을 Raw 264.7 세포에 처리한 후 MTT assay로 측정한 결과 고농도 $100{\mu}g/mL$까지 세포독성은 없었으며, NO 및 TNF-${\alpha}$의 분비를 농도 유의적으로 증가시켰다. 또한 AGS 위암 세포 성장저해효과를 확인하기 위하여 MTT assay를 하였고 그 결과 농도와 시간 의존적으로 암 세포 성장이 유의적으로 감소하였다. Apoptosis로 인해 암 세포 성장이 감소하였는지 확인하기 위하여 DAPI 염색을 한 결과 apoptotic body와 세포질 응축이 시간 의존적으로 증가하는 것을 확인하였다. 또한 fucoidan은 미토콘드리아의 투과율을 향상시키며 미토콘드리아에서 방출되는 cytochrome c의 발현을 증가시켰다. Western blotting의 결과 시간 의존적으로 anti-apoptotic 분자인 Bcl-2와 XIAP 발현 감소와 반대로 pro-apoptotic 분자인 Bax 발현이 증가하였다. Cleaved-caspase-9의 발현이 증가하였으며 Akt의 인산화는 시간 의존적으로 감소하였다. Caspase 억제제인 z-VAD-FMK 처리 시 Bax, caspase-9의 발현을 감소시켜 apoptosis 유도를 억제하였으며 이러한 결과는 caspase가 apoptosis 유도에 중요한 역할을 하는 것으로 나타낸다. 본 실험의 AGS 위암 세포주에서 대조군에 비하여 fucoidan 처리군에서 면역세포 활성 및 AGS 위암 세포주에서 caspase 활성을 통해 apoptosis를 유도하는 것으로 사료된다.

Keywords

References

  1. Risch, H.A., Jain, M., Choi, N.W., Fodor, J.G., Pfeiffer, C.J., Howe, G.R., Harrison, L.W., Craib, K.J. and Miller, A.B. : Dietary factors and the incidence of cancer of the stomach. Am. J. Epidemiol, 122, 947-959 (1985).
  2. Lee, K.H., Kown, H.J., Chun, S.S., Kim, J.H., Cho, Y.J. and Cha, W.S. : Biological activities of extracts from phellinus linteus. J. Korean. Soc. Appl. Biol. Chem, 49, 298-303 (2006).
  3. Kim, S.W. and Kim, E.S. : Studies on the immunomodulating effect of polysaccharide extracted from Ganoderma lucidum on machrophage. J. Korean. Soc. Food. Sci. Nutr, 26, 148-153 (1997).
  4. Lee, M.K., Choi, G.P., Ryu, L.H., Lee, G.Y., Yu, C.Y. and Lee, H.Y. : Enhanced immune activity and cytotoxicity of Artemisia capillaris Thub. extracts against human cell lines. Korean. J. Med. Crop. Sci, 12, 36-42 (2004).
  5. Schaeffer, D.J. and Krylov, V.S. : Anti-HIV activity of extracts and compounds from algae and cyanobacteria, Ecotoxicol. Environ. Saf, 45, 208-227 (2000). https://doi.org/10.1006/eesa.1999.1862
  6. Dobashi, K., Nishino, T., Fujihara, M., and Nagumo, T. : Isolation and preliminary characterization of fucose-containing sulfated polysaccharides with blood -anticoagulant activity from the brown seaweed Hizikia fusiforme. Carbohydr. Res, 194, 315-320 (1989). https://doi.org/10.1016/0008-6215(89)85032-3
  7. Nishino, T., Nishioka, C., Ura, H. and Nagumo, T. : Isolation and partial characterization of a novel amino sugar-containing fucan vesiculosus fucoidan. Carbohydr. Res, 255, 213-214 (1994). https://doi.org/10.1016/S0008-6215(00)90980-7
  8. Aisa, Y., Miyakawa, Y., Nakazato, T., Shibata, H., Saito, K., Ikeda, Y. and Kizaki, M. : Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways. Am. J. Hematol, 78, 7-14 (2005). https://doi.org/10.1002/ajh.20182
  9. Hyun, J.H., Kimg, S.C., Kang, J.I., Kim, M.K., Boo, H.J., Kwon, J.M., Koh, Y.S., Hyun, J.W., Park, D.B., Yoo, E.S. and Kang, H.K. : Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells. Biol. Pharm. Bull, 32, 1760-1764 (2009). https://doi.org/10.1248/bpb.32.1760
  10. Nagamine, T., Hayakawa, K., Kusakabe, T., Takada, H., Nakazato, K., Hisanaga, E. and Iha, M. : Inhibitory effect of fucoidan on Huh 7 hepatoma cells through down-regulation of CXCL12. Nutr. Cancer, 61, 340-347 (2009). https://doi.org/10.1080/01635580802567133
  11. Yamasaki-Miyamoto, Y., Yamasaki, M., Tachibana, Ha. and Yamada, K. : Fucoidan induces apoptosis through activation of caspase-8 on human breast cancer MCF-7 cells. J. Agric. Food. Chem, 57, 8677-8682 (2009). https://doi.org/10.1021/jf9010406
  12. Ko, E.J. and Joo, H.G. : Immunostimulatory effects of fucoidan on mouse splenocytes. Lab. Anim. Res, 25, 195-200 (2009).
  13. Yang, J.W., Yoon, S.Y., Oh, S.J., Kim, S.K. and Kang, K.W. : Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase. Biochem. Biophys. Res. Commun, 346, 345-350 (2006). https://doi.org/10.1016/j.bbrc.2006.05.135
  14. Maruyama, H., Tamauchi, H., Iizuka, Ma. and Nakano, T. : The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls. Planta. Med, 72, 1415-1417 (2006). https://doi.org/10.1055/s-2006-951703
  15. Kim, N.H. and Joo, H.G.: Immunostimulator effects of fucoidan on bone marrow-derived dendritic cells. Immunol. Lett, 115, 138-143 (2008). https://doi.org/10.1016/j.imlet.2007.10.016
  16. Hengrtner, M.O. : The biochemistry of apoptosis. Nature, 407, 770-777 (2000). https://doi.org/10.1038/35037710
  17. Hamsa, T.P. and Kuttan, G. : Evalution of the anti-inflammatory and anti-tumor effect of ipomoea obscura(L) and its mode of action through the inhibition of proinflammatory cytokines, nitric oxide and COX-2. Inflammation, 34, 171-183 (2011). https://doi.org/10.1007/s10753-010-9221-4
  18. Kang, N.S. and Sohn, E.H. : Immunomodulatory effects of fructus and semen from Rosa rugosa on macrophages. J. Korean. Plant. Res, 23, 399-405 (2010).
  19. Zhang, L., Zhu, Y., Lun, D., Yu, B. and Zhu, X. : Proteomic analysis of macrophages: a potential way to identify novel proteins associated with activation of macrophages for tumor cell killing. Cell. Mol. Immunol, 4, 359-367 (2007).
  20. Bodmer, J.L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J. and Tschopp, J. : TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat. Cell. Biol, 2, 241-243 (2000). https://doi.org/10.1038/35008667
  21. Kischkel, F.C., Lawrence, D.A., Chuntharapai, A., Schow, P., Kim, K.J. and Ashkenazi, A. : Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity, 12, 611-620 (2000). https://doi.org/10.1016/S1074-7613(00)80212-5
  22. Cohen, G.M. : Caspase: the executioners of apoptosis. Biochem. J, 326, 1-16 (1997).
  23. Li, H., Zhu, H., Xu, C.J. and Yuan, J. : Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 94, 491-501 (1998). https://doi.org/10.1016/S0092-8674(00)81590-1
  24. Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmever, D.D., Wang, H.G., Reed, J.C., Nicholson, D.W., Alnemri, E.S., Green, D.R. and Martin, S.J. : Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspase-2, -3, -6, -7, -8 and -10 in a caspase-9 dependent manner. J. Cell. Biol, 144, 281-292 (1999). https://doi.org/10.1083/jcb.144.2.281
  25. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D. and Korsmeyer, S.J. : Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature, 348, 334-336 (1990) https://doi.org/10.1038/348334a0
  26. Deveraux, Q.L. and Reed, J.C. : IAP family proteins-suppressors of apoptosis. Genes. Dev, 13, 1899-1911 (1999) https://doi.org/10.1101/gad.13.15.1899
  27. Carnero, A. : The PKB/AKT pathway in cancer. Curr. Pharm, 277, 34-44 (2010)
  28. Osaki, M., Oshimura, M. and Ito, H. : PI3K-Akt pathway: its functions and altherations in human cancer. Apoptosis, 9, 667-676 (2004) https://doi.org/10.1023/B:APPT.0000045801.15585.dd
  29. Kim, E.J., Park, S.Y., Lee, J.Y. and Park, J.H. : Fucoidan present in brown algae induces apoptosis of human colon cancer cells. BMC. Gastroenterol, 10, 96-107 (2010) https://doi.org/10.1186/1471-230X-10-96
  30. Jung, J.I., Lim, S.S., Choi, H.J., Cho, H.J., Shin, H.K., Kim, E.J., Chung, W.Y., Park, K.K. and Park, J.H. : Iosliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells. J. Nutr. Biochem, 17, 689-696 (2006). https://doi.org/10.1016/j.jnutbio.2005.11.006
  31. Palmer, R.M., Ferrige, A.G. and Moncada, S. : Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature, 327, 524-526 (1987) https://doi.org/10.1038/327524a0
  32. Moncada, S. and Higgs, A. : The L-arginine nitric oxide pathway. N. Engl. J. Med, 329, 2002-2012 (1993) https://doi.org/10.1056/NEJM199312303292706
  33. Yang, W.W. and Krukoff, T.L. : Nitric oxide regulates body temperature, neuronal activation and interleukin-1${\beta}$ gene expression in the hypothalamic paraventricular nucleus in response to immune stress. Neuropharmacology, 39, 2075-2089 (2000). https://doi.org/10.1016/S0028-3908(00)00054-X
  34. Hang, D., Choi, H.S., Kang, S.C., Kim, K.R., Sohn, E.S., Kim, M.H., Pyo, S. and Son, E. : Effects of fucoidan on NO production and phagocytosis of macrophages and the proliferation of neuron cells. J. Food. Sci. Nutr, 10, 344-348 (2005). https://doi.org/10.3746/jfn.2005.10.4.344
  35. Strieter, R., Kunkel, S. and Bone, R. : Role of tumor necrosis factor-alpha in disease states and inflammation. Crit. Car. Med, 21, 447-463 (1993). https://doi.org/10.1097/00003246-199303000-00024
  36. Adams, D.O. and Hamilton, T.A. : The cell biology of macrophage activation. Annu. Rev. Immunol, 2, 283-318 (1984). https://doi.org/10.1146/annurev.iy.02.040184.001435
  37. Nathan, C.F. : Secretory products of macrophages. J. Clin. Invest, 79, 319-326 (1987). https://doi.org/10.1172/JCI112815
  38. Vishvakarma, N.K. and Slingh, S.M. : Immunopotentiating effect of proton pump inhibitor pantoprazole in a lymphoma-bearing murine host: Implication in antitumor activation of tumorassociated macrophages. Immunol. Lett, 134, 823-92 (2010).
  39. Evans, V.G. : Multiple pathways to apoptosis. Cell. Biol. Int, 17, 461-476 (1993). https://doi.org/10.1006/cbir.1993.1087
  40. Donovan, M. and Cotter, T.G. : Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. Biochim. Biophys. Acta, 1644, 133-147 (2004). https://doi.org/10.1016/j.bbamcr.2003.08.011
  41. Rosse, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B. and Bomer, C. : Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature, 391, 496-499 (1998). https://doi.org/10.1038/35160
  42. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. and Wang, X. : Cytochrome c and ATPdependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 91, 479-489 (1997). https://doi.org/10.1016/S0092-8674(00)80434-1
  43. Du, C., Fang, M., Li, Y., Li, L. and Wang, X. : Smac, a mitochondrial protein that promotes cytochrom c-dependent caspase activation by eliminating IAP inhibition. Cell, 102, 33-42 (2002).
  44. Yoeli-Lemer, M., Yiu, G.K., Rabinovitz, I., Erhardt, P., Jauliac, S. and Toker, A. : Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT. Mol. Cell, 20, 539-550 (2005). https://doi.org/10.1016/j.molcel.2005.10.033
  45. Okoshi, R.T., Ozaki, H., Yamamoto, K., Ando, N., Koida, S., Ono, T., Koda, T., Kamijo, A., Nakagawara, A. and Kizaki, H.: Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress. J. Biol. Chem, 283, 3979-3987 (2008). https://doi.org/10.1074/jbc.M705232200