AN EXPERIMENTAL STUDY ON MISTLETOE EXTRACT-INDUCED APOPTOSIS IN ORAL SQUAMOUS CELL CARCINOMA

구강편평세포암종에서 미슬토 추출물의 Apoptosis 유도 효과에 대한 실험적 연구

  • Heo, Gyun-Haeng (Department of Oral & Maxillofacial Surgery, College of Dentistry, Dankook University) ;
  • Lee, Jae-Hoon (Department of Oral & Maxillofacial Surgery, College of Dentistry, Dankook University) ;
  • Kim, Chul-Hwan (Department of Oral & Maxillofacial Surgery, College of Dentistry, Dankook University)
  • 허균행 (단국대학교 치과대학 구강악안면외과학교실) ;
  • 이재훈 (단국대학교 치과대학 구강악안면외과학교실) ;
  • 김철환 (단국대학교 치과대학 구강악안면외과학교실)
  • Published : 2005.02.28

Abstract

This study was performed to investigate mistletoe extract-induced apoptosis in oral squamous cell carcinoma. In vivo study, HN22 cells were xenografted in nude mice. After tumor was experimentally induced, mistletoe extract was directly injected on the tumor mass. The specimens were evaluated using light and transmission electron microscopes. In vitro study, HN22 cells were cultured and exposed to mistletoe extract. The cells were evaluated using transmissin electron microscope. To evaluate apoptotic cells, flow cytometric analysis was done. The results were obtained as follows: 1. Light microscopic view of tumor mass showed necrosis at 2-4 weeks. 2. Transmission electron micrographs of tumor mass showed apoptosis and necrosis. 3. In TEM view of cell lines, necrosis and apoptosis were shown with mistletoe extract at $300{\mu}g/ml$, apoptosis was shown with mistletoe extract at $100{\mu}g/ml$. 4. In flow cytometric analysis, early and late apoptosis was shown when using caspase-3Ab and annexin-V, but no significant change was noted when using mebstain and Apo2.7 Ab. In this study, mistletoe extract induced necrosis and apoptosis in the tumor mass was induced by HN22 cells, early and late apoptosis in vitro study. Mistletoe extract was likely to induce cell death in oral squamous cell carcinoma through apoptosis.

Keywords

References

  1. Mansky PJ: Mistletoe and Cancer: controversies and perspectives. Seminar in Oncology Dec 2002;29(6):589-594
  2. Kovac S, Hajto T, Hostanska K: Improvement of DNA repair in lymphocytes of breast cancer patient treated with viscum album extract(Iscador). Eur J Cancer 1991;27:1672-1676 https://doi.org/10.1016/0277-5379(91)90443-H
  3. Bussing A: Mistletoe: A story with an open end. Anticancer Drugs 1997;8(suppl.1):1-2
  4. Frauz H, Ziska P, kindt A: Isolation and properties of three lectin from misteletoe(Viscum album L.). J Biochem 1981;195:481-484
  5. Peumans WJ, Verhaert P, Pf?ller U, et al.: Isolation and partial characterization of a small chintin-binding lection from mistletoe (Viscum-album). FEBS Lett 1996;396:261-265 https://doi.org/10.1016/0014-5793(96)01108-8
  6. Schaller G, Urech K: Cytotoxicity and different viscotoxins and extracts from the europeans subspecies of Viscum album L. phytotheraphy Research 1996;10:473-477 https://doi.org/10.1002/(SICI)1099-1573(199609)10:6<473::AID-PTR879>3.0.CO;2-Q
  7. Bocci V: Mistletoe(Viscum album) lectins as cytokine inducers and immunoadjuvant in tumor therapy. A review. J Biol Reg Homeos Agent 1993;7:1-6
  8. Franz H: Mistletoe lectins and their A and B chains. Oncology 1986;5 43:23-24 https://doi.org/10.1159/000214961
  9. Burger AM, Mengs U, Schuler JB, et al.: Antiproliferative activity of an aqueous mistletoe extract in human tumor cell line and xenografts in vitro. Drug Res 2001;51:748-757
  10. Yokoda J: Tumor progression and metastasis. Carcinogenesis 2000;21:497-503 https://doi.org/10.1093/carcin/21.3.497
  11. Kutan SAR, Kuttan G: Effect of Viscum album in the inhibition of lung metasis in mice induced by B 6F10 melanoma cells. J Exp Clin Cancer Res 1999;6:159-162
  12. Folkman J: Tumour angiogenesis. Adv Cancer Res 1985;43:175-203 https://doi.org/10.1016/S0065-230X(08)60946-X
  13. Forkman J, Klagsbrun M: Angiogenic factors. Science 1987;235:442- 447 https://doi.org/10.1126/science.2432664
  14. Yoon TJ, Yoo YC, Choi OB, et al.: Inhibitory effect of Korean mistletoe(Viscum album coloratum) extract on tumor angiogenesis and metastasis of haematogenous and non-haematogenous tumor cells in mice. Cancer Lett 1995;97:83-91 https://doi.org/10.1016/0304-3835(95)03956-W
  15. Stein GM, Berg PA: Mistletoe extract-Induced effects on Immunocompetent cells : In vitro study. Anticancer Drugs(Suppl 1): 1997;s39-42
  16. Park JH, Hyun CK, Shin HK: Cytotoxic effects of the components in heat-treated mistletoe (Viscum album). Cancer Lett 1999;139:207- 213 https://doi.org/10.1016/S0304-3835(99)00043-9
  17. Stein GM, Schaller G, Pfuller U: Thionins from Viscum album L : Influence of the viscotoxins on the activation of granulocytes. Anticancer Res 1999;19:1037-1042
  18. Fillis RE, Yuan J, Horvitz HR: Mechanism and functions of cell death. Ann Rev Cell Biol 1991;7:663-698 https://doi.org/10.1146/annurev.cb.07.110191.003311
  19. Krammer PH, Behnnann I, Daniel P, et al.: Regulation of apoptosis in the immune system. Curr Biol 1994;6:279-289 https://doi.org/10.1016/S0960-9822(02)00478-5
  20. Jassen O, Scheffler A, Kabelitz D: In vitro effect of mistletoe extracts and mistletoe lectins. Drug Res 1993;43(11):1221-1227
  21. Kimura C, Kondo OL, Amatsu M, et al.: Mechanism of UV-induced apoptosis in Human leukemia cells: roles of Ca2+/Mg2+ endonuclease, caspase-3, and stress-activated protein kinase. Exp Cell Res 1998;239:411-422 https://doi.org/10.1006/excr.1997.3912
  22. Schewartz LM, Osborn BA: Programmed cell death, apoptosis and killer genes. Immunol Today 1993;14:582-590 https://doi.org/10.1016/0167-5699(93)90197-S
  23. Kerr JFR, Winterford CM, Harmon BV: Apoptosis-its significance in cancer and cancer therapy. Cancer 1994;73:2013-2026 https://doi.org/10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J
  24. Wallance BRR, lowe SW: Clinical implications of mutations. Cell Mol Life Sci 1999;55:64-75 https://doi.org/10.1007/s000180050270
  25. Yonish-Rouach E, Resnizky D, Lotem J, et al.: Wild-type p53 induces apoptosis of myeloid leukemic cells that is inhibited by interleukin- 6. Nature 1991;352:345-347 https://doi.org/10.1038/352345a0
  26. Lowe SW, Lin AW: Apoptosis in cancer. Carcinogenesis 2000; 21:485-495 https://doi.org/10.1093/carcin/21.3.485
  27. Tereda N, Lucas JJ, Gelfand EW: Differential regulation of tumor suppressor molecules, retinoblastoma susceptibility gene product (Rb) and p53, during cell cycle progression of normal human T cells. J Immunol 1991;147:698-704
  28. Kuirbitz SJ, Plunkett BS, Walsh WV, et al.: Wild type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA 1992;89:7491-7495
  29. El-Deiry WS, Harper JW, OConner PM, et al.: WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994;54:1169-1174
  30. Jiang H, Lin J, Su Z, Collart FR, et al.: Induction of Differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1 expression in the abscence of p53. Oncogene 1994;9:3397-3406
  31. Kano A, Watanabe Y, Takeda N, et al.: Analysis of IFN-gammainduced cell cycle arrest and cell death in hepatocytes. J Biochem 1997;121:677-683 https://doi.org/10.1093/oxfordjournals.jbchem.a021639
  32. Kim WH, Kang KH, Kim MY, et al.: Induction of p53-independent p21 during ceramide-induced G1 arrest in human hepatocarcinoma cells. Biochem Cell Biol 2000;78:127-135 https://doi.org/10.1139/bcb-78-2-127
  33. Antonsson B, Martinou JC: The Bcl-2 protein family. Exp Cell Res 2000;256:50-57 https://doi.org/10.1006/excr.2000.4839
  34. Hofmann B, Reed JC: Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 1994;9:1799- 1805
  35. Kim YS, Jin SH, Lee YH, et al.: Ginsenoside Rh2 induces apoptosis independently of Bcl-2, Bcl-XL or Bax in C6Bu-1 cells. Arc Pharm Res 1999;22:448-453 https://doi.org/10.1007/BF02979151
  36. Kelekar A, Thompson CB: Bcl-2 family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 1998;8:324-330 https://doi.org/10.1016/S0962-8924(98)01321-X
  37. Boise LH, Gonzalez-Garcia M, Postema CE, et al.: Bcl-xL, a bcl-2- related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993;74:597-608 https://doi.org/10.1016/0092-8674(93)90508-N
  38. Hodes R: Molecular targeting of cancer : Telomeres as targets. Proc Natl Acad Sci USA 2001;98:7649-7651
  39. Liu JP: Studies on the molecular mechanisms in the regulation of telomerase activity. FASEB J 1999;13:2091-2104
  40. Lyu SY, Park WB, Choi KH, et al.: involvement of caspase-3 in apoptosis induced by Viscum album var. coloratum agglutinin in HL- 60 cells. Biosci Biotechnol Biochem 2001;65(3):534-541 https://doi.org/10.1271/bbb.65.534
  41. Martin SJ, Green DR: Protease activation during apoptosis : death by a thousand cuts. Cell 1995;82:349-352 https://doi.org/10.1016/0092-8674(95)90422-0
  42. Emoto Y, Manome Y, Meinhardt G, et al.: Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells. EMBO J 1995;14:6148-6156
  43. Ghayur T, Hugunin M, Talanian RV, et al.: Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis. J Exp Med 1996;184:2399-2404 https://doi.org/10.1084/jem.184.6.2399
  44. Khwaja A, Tatton L: Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis. Blood 1999;94:291-301
  45. Alnemri ES, Livingston DJ, Nicholson DW, et al.: Human ICE/CED- 3 protease nomenclature(letter). Cell 1996;87:171 https://doi.org/10.1016/S0092-8674(00)81334-3
  46. Schulze-Osthoff K, Ferrari D, Los M, et al.: Apoptosis signaling by death receptors. Eur J Biochem 1998;254:439-459 https://doi.org/10.1046/j.1432-1327.1998.2540439.x
  47. Kroemer G, Zamzami N, Susin SA: Mitochondrial control of apoptosis. Immunol Today 1997;18:44-51 https://doi.org/10.1016/S0167-5699(97)80014-X
  48. Lizard GL, Fournel S, Genestier L, et al.: Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis. Cytometry 1995;21:275-283 https://doi.org/10.1002/cyto.990210308
  49. Majno G, Joris I: Apoptosis, oncosis, and necrosis, an overview of cell death. Am J Pathol 1995;146:3-15
  50. Bussing A, Wagner M, Wagner B, et al.: Induction of mitochondrial Apo 2.7 molecules and generation of reactive oxygen-intermediates cultured lymphocytes by the toxic protein from Viscum album L. Cancer Lett 1999;139:79-88 https://doi.org/10.1016/S0304-3835(99)00010-5
  51. Koester SK, Roth P, Mikulka WR, et al.: Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein specific monoclonal antibody Apo 2.7. Cytometry 1997;29:306- 312 https://doi.org/10.1002/(SICI)1097-0320(19971201)29:4<306::AID-CYTO7>3.0.CO;2-7
  52. Zhang C, Ao Z, Seth A, et al.: Mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells. J Immunol 1996;157:3980-3987
  53. Martin SJ: Protein or RNA synthesis inhibiton induces apoptosis of mature human CD4+ T cell blasts. Immunol Lett 1993;35:125-134 https://doi.org/10.1016/0165-2478(93)90080-L
  54. Overbeeke R, Steffens-Nakken H, Vermes I, et al.: Early feature of apoptosis detected by four different cytometric assay. Apoptosis 1998;3:11.5-121 https://doi.org/10.1023/A:1009649025439