Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Apoptosis of Human Hepatocarcinoma (HepG2) and Neuroblastoma (SK-N-SH) Cells Induced by Polysaccharides-Peptide Complexes Produced by Submerged Mycelial Culture of an Entomopathogenic Fungus Cordyceps sphecocephala

  • Published : 2008.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Three different polysaccharide-peptide complexes (PPC, named as Fr-I, Fr-II, and Fr-III) were produced by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala, and their anticancer activities were investigated in human hepatocarcinoma (HepG2) and neuroblastoma (SK-N-SH) cells. The highest inhibitory effects of PPC on both HepG2 and SK-N-SH cells were achieved with Fr-I, whereas Fr-III with low molecular mass showed lower inhibition effects. Interestingly, the inhibitory effects of the three fractions were increased after protease digestion, suggesting that the inhibitory effects resulted mainly from the carbohydrate moiety, at least in the case of Fr-II and Fr-III, of PPC. The results of DNA fragmentation in PPC-induced apoptotic cells were confirmed by both DNA ladder assay and comet assay. Our investigation also showed that PPC-induced apoptosis of both cancer cells was associated with intracellular events including DNA fragmentation, activation of caspase-3, and modulation of Bcl-2 and Bax. We conclude that PPC has potential as a novel therapeutic agent for the treatment of both HepG2 and SK-N-SH cancer cells without any cytotoxicity against normal cells.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Budihardjo, I., H. Oliver, M. Lutter, X. Luo, and X. Wang. 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15: 269-290 https://doi.org/10.1146/annurev.cellbio.15.1.269
  3. Chen, Y. J., M. S. Shiao, S. S. Lee, and S. Y. Wang. 1997. Effects of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells. Life Sci. 60: 2349-2359 https://doi.org/10.1016/S0024-3205(97)00291-9
  4. Choi, D. B., J. M. Maeng, J. L. Ding, and W. S. Cha. 2007. Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola. J. Microbiol. Biotechnol. 17: 1369-1378
  5. Compton, M. M. 1992. A biochemical hallmark of apoptosis: Internucleosomal degradation of the genome. Cancer Met. Rev. 11: 105-119 https://doi.org/10.1007/BF00048058
  6. Cooper, J. D., T. M. Michaelidis, G. Z. Tzimagiorgis, M. Sendtner, M. Meyer, and H. Thoenen. 1996. Cholinergic neuron-specific overexpression of Bax in ChAT Bax transgenic mice. Soc. Neurosci. 22: 570
  7. Earnshaw, W. C. 1995. Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7: 337-343 https://doi.org/10.1016/0955-0674(95)80088-3
  8. Evan, G. I. and K. H. Vousden. 2001. Proliferation, cell cycle and apoptosis in cancer. Nature 411: 342-348 https://doi.org/10.1038/35077213
  9. Fukamachi, Y., Y. Karasaki, T. Sugiura, H. Itoh, T. Abe, K. Yamamura, and K. Higashi. 1998. Zinc suppresses apoptosis of U937 cells induced by hydrogen peroxide through an increase of the Bcl-2/Bax ratio. Biochem. Biophys. Res. Commun. 246: 364-369 https://doi.org/10.1006/bbrc.1998.8621
  10. Gorczyca, W., K. Bigman, A. Mittelman, T. Ahmed, J. Gong, M. R. Melamed, and Z. Darzynkiewicz. 1993. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7: 659-670
  11. Jeong, S. C., B. K. Yang, K. S. Ra, M. A. Wilson, Y. Cho, Y. A. Gu, and C. H. Song. 2004. Characteristics of anticomplementary biopolymer extracted from Coriolus versicolor. Carbohydr. Polym. 55: 255-263 https://doi.org/10.1016/j.carbpol.2003.09.012
  12. Kaufmann, S. H. and W. C. Earnshaw. 2000. Induction of apoptosis by cancer chemotherapy. Exp. Cell Res. 256: 42-49 https://doi.org/10.1006/excr.2000.4838
  13. Kiho, T., A. Yamane, J. Hui, S. Usui, and S. Ukai. 1996. Polysaccharides in fungi XXXVI: Hypoglycemic activity of a polysaccharide (CS-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Biol. Pharm. Bull. 19: 294-296 https://doi.org/10.1248/bpb.19.294
  14. Kiho, T., K. Ookubo, S. Usui, S. Ukai, and K. Hirano. 1999. Strutural features and hypoglycemic activity of a polysaccharide (CS-F10) from the cultured mycelium of Cordyceps sinensis. Biol. Pharm. Bull. 22: 966-970 https://doi.org/10.1248/bpb.22.966
  15. Kim, H. M., S. W. Kim, H. J. Hwang, M. K. Park, Y. A. G. Mahmoud, J. W. Choi, and J. W. Yun. 2006. Influence of agitation intensity and aeration rate on the production of antioxidative exopolysaccharides from submerged mycelial culture of Ganoderma recinaceum. J. Microbiol. Biotechnol. 16: 1240-1247
  16. Kim, S. W., H. J. Hwang, C. P. Xu, J. M. Sung, J. W. Choi, and J. W. Yun. 2003. Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738. J. Appl. Microbiol. 94: 120-126
  17. Kobayashi, T., S. Ruan, K. Clodi, K. O. Kliche, H. Shiku, M. Andreeff, and W. Zhang. 1998. Overexpression of Bax gene sensitizes K562 erythroleukemia cells to apoptosis induced by selective chemotherapeutic agents. Oncogene 16: 1587-1591 https://doi.org/10.1038/sj.onc.1201681
  18. Koh, J. H., J. M. Kim, U. J. Chang, and H. J. Suh. 2003. Hypocholesterolemic effect of hot-water extract from mycelia of Cordyceps sinensis. Biol. Pharm. Bull. 26: 84-87 https://doi.org/10.1248/bpb.26.84
  19. Kumar, S., P. K. Suresh, M. R. Vijayababu, A. Arunkumar, and J. Arunakaran. 2006. Anticancer effects of ethanolic neem leaf extract on prostate cancer cell line (PC-3). J. Ethnopharmacol. 105: 246-250 https://doi.org/10.1016/j.jep.2005.11.006
  20. Lazebnik, Y. A., S. H. Kaufmann, S. Desnoyers, G. G. Poirier, and W. C. Earnshaw. 1994. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371: 346-347 https://doi.org/10.1038/371346a0
  21. Li, N., X. H. Liu, J. Zhou, Y. X. Li, and M. W. Zhao. 2006. Analysis of influence of environmental conditions on ganoderic acid content in Ganoderma lucidum using orthogonal design. J. Microbiol. Biotechnol. 16: 1940-1946
  22. Lim, H. Y., Y. H. Lim, Y. H. Cho, and C. H. Lee. 2006. Induction of apoptosis in the HepG2 cells by HY53, a novel natural compound isolated from Bauhinia forficata. J. Microbiol. Biotechnol. 16: 1262-1268
  23. Liu, F., V. E. C. Ooi, and S. T. Chang. 1995. Anti-tumour components of the culture filtrates from Tricholoma sp. World J. Microbiol. Biotechnol. 11: 486-490 https://doi.org/10.1007/BF00286357
  24. Martin, S. J. and D. R. Green. 1995. Protease activation during apoptosis: Death by a thousand cuts? Cell 82: 349-352 https://doi.org/10.1016/0092-8674(95)90422-0
  25. Matsunaga, K., A. Hosokawa, M. Oohara, N. Sugita, M. Harada, and K. Nomoto. 1998. Direct action of a proteinbound polysaccharide, PSK, on transforming growth factor-$\beta$. Immunopharmacology 40: 219-230 https://doi.org/10.1016/S0162-3109(98)00045-9
  26. Mizuno, T., P. Yeohlui, T. Kinoshita, C. Zhung, H. Ito, and Y. Mayuzumi. 1996. Antitumor activity and chemical modification of polysaccharides from Nioshimeiji mushroom, Tricholoma giganteum. Biosci. Biotechnol. Biochem. 60: 30-33 https://doi.org/10.1271/bbb.60.30
  27. Moradali, M. F., H. Mostafavi, S. Ghods, and G. A. Hedjaroude. 2007. Immunomodulating and anticancer agents in the realm of macromycetes fungi (macrofungi). Int. Immunopharmacol. 7: 701-724 https://doi.org/10.1016/j.intimp.2007.01.008
  28. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  29. Nakamura, K., Y. Yamaguchi, S. Kagota, K. Shinozuka, and M. Kunitomo. 1999. Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats. Jap. J. Pharmacol. 79: 505-508 https://doi.org/10.1254/jjp.79.505
  30. Oh, J. Y., E. J. Cho, S. H. Nam, J. W. Choi, and J. W. Yun. 2007. Production of polysaccharide-peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala. Process Biochem. 42: 3352-3362
  31. Ohmori, T., K. Tamura, K. Fukui, G. Kawanishi, M. Mitsuyama, K. Nomoto, and T. Miyazaki. 1989. Isolation of galactosaminoglycan moiety (CO-N) from protein-bound polysaccharides of Cordyceps ophioglossoides and its effects against murine tumors. Chem. Pharm. Bull 37: 1019-1022 https://doi.org/10.1248/cpb.37.1019
  32. Oltvai, Z. N., C. L. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74: 609-619 https://doi.org/10.1016/0092-8674(93)90509-O
  33. Park, J. P., Y. M. Kim, S. W. Kim, H. J. Hwang, Y. J. Cho, Y. S. Lee, and J. W. Yun. 2002. Effect of aeration rate on the mycelial morphology and exo-biopolymer production in Cordyceps militaris. Process Biochem. 37: 1257-1262 https://doi.org/10.1016/S0032-9592(02)00005-5
  34. Peng, Y., L. Zhang, F. Zeng, and Y. Xu. 2003. Structure and antitumor activity of extracellular polysaccharides from mycelium. Carbohydr. Polym. 54: 297-303 https://doi.org/10.1016/S0144-8617(03)00190-5
  35. Sakahira, H., M. Enari, and S. Nagata. 1998. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391: 96-99 https://doi.org/10.1038/34214
  36. Salvesen, G. S. and V. M. Dixit. 1997. Caspases: Intracellular signaling by proteolysis. Cell 91: 443-446 https://doi.org/10.1016/S0092-8674(00)80430-4
  37. Surenjav, U., L. Zhang, X. Xu, X. Zhang, and F. Zeng. 2006. Effects of molecular structure on antitumor activities of (13)-$\beta$-D-glucans from different Lentinus edodes. Carbohydr. Polym. 63: 97-104 https://doi.org/10.1016/j.carbpol.2005.08.011
  38. Tao, Y., L. Zhang, and P. C. K. Cheung. 2006. Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides. Carbohydr. Res. 341: 2261-2269 https://doi.org/10.1016/j.carres.2006.05.024
  39. Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. M. Pomares, S. Gordon, and S. Y. C. Wong. 2002. The $\beta$-glucan receptor, Dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169: 3876-3882 https://doi.org/10.4049/jimmunol.169.7.3876
  40. Thornberry, N. A. 1997. The caspase family of cysteine proteases. Br. Med. Bull. 53: 478-490 https://doi.org/10.1093/oxfordjournals.bmb.a011625
  41. Tice, R. R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu, and Y. F. Sasaki. 2000. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206-221 https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
  42. Tu, H., S. C. Jacobs, A. Borkowski, and N. Kyprianou. 1996. Incidence of apoptosis and cell proliferation in prostate cancer: Relationship with TGF-b and bcl-2 expression. Int. J. Cancer 69: 357-363 https://doi.org/10.1002/(SICI)1097-0215(19961021)69:5<357::AID-IJC1>3.0.CO;2-4
  43. Wang, H. X., T. B. Ng, V. E. Ooi, W. K. Liu, and S. T. Chang. 1996. A polysaccharide-peptide complex from cultured mycelia of the mushroom Tricholoma mongolicum with immunoenhancing and anti-tumor activities. Biochem. Cell Biol. 71: 95-100
  44. Wasser, S. P. 2002. Medicinal mushrooms as a source of antitumor and immuno-modulating polysaccharides. Appl. Microbiol. Biotechnol. 60: 258-274 https://doi.org/10.1007/s00253-002-1076-7
  45. Yamada, H., H. Kiyohara, J. C. Cyong, and Y. Otsuka. 1985. Studies on the polysaccharides from Angelica acutiloba. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitakawa. Mol. Immunol. 22: 295-302 https://doi.org/10.1016/0161-5890(85)90165-8
  46. Yamada, H., K. Ohtani, H. Kiyohara, J. C. Cyung, Y. Otsuka, Y. Ueno, and S. Omura. 1985. Purification and chemical properties of anti-complementary polysaccharide from the leaves of Artemisia princeps. Planta Medica 51: 121-125 https://doi.org/10.1055/s-2007-969424
  47. Yasuhara, S., Y. Zhu, T. Matsui, N. Tipirneni, Y. Yasuhara, M. Kaneki, A. Rosenzweig, and J. A. Martyn. 2003. Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis. J. Histochem. Cytochem. 51: 873-885 https://doi.org/10.1177/002215540305100703
  48. Yin, X. M., Z. Oltvai, and S. J. Korsmeyer. 1994. BH1 and BH2 domains of bcl-2 are required for inhibition of apoptosis and heterodimerization with bax. Nature 369: 321-323 https://doi.org/10.1038/369321a0
  49. Zhang, M., S. W. Cui, P. C. K. Cheung, and Q. Wang. 2007. Antitumor polysaccharides from mushrooms: A review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci. Technol. 18: 4-19 https://doi.org/10.1016/j.tifs.2006.07.013
  50. Zhu, T., L. Collins, J. Kelly, Z. J. Xiao, S. H. Kim, and C. Y. Chen. 2007. Phellinus linteus activates different pathways to induce apoptosis in prostate cancer cells. Br. J. Cancer 96: 583-590 https://doi.org/10.1038/sj.bjc.6603595