Response of Metastatic Cancer Cells to Thermal Changes in vitro

배양온도 변화에 대한 전이성 암세포의 반응

  • Ahn, San-Gil (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University) ;
  • Kwon, Young-Ee (College of Pharmacy, Woosuk University) ;
  • Choi, Ho-Soon (Department of Internal Medicine, College of Medicine, Hanyang University) ;
  • Kwon, Jung-Kyun (Electron Microscopy Laboratory, College of Medicine, Hanyang University) ;
  • Yoo, Jin-Young (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University) ;
  • Kim, Jong-Ryong (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University) ;
  • Kim, Won-Kyu (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University)
  • 안상길 (한양대학교 의과대학 해부세포생물학교실) ;
  • 권영이 (우석대학교 약학대학) ;
  • 최호순 (한양대학교 의과대학 내과학교실) ;
  • 권중균 (한양대학교 의과대학 전자현미경실) ;
  • 유진영 (한양대학교 의과대학 해부세포생물학교실) ;
  • 김종룡 (한양대학교 의과대학 해부세포생물학교실) ;
  • 김원규 (한양대학교 의과대학 해부세포생물학교실)
  • Published : 2007.12.31

Abstract

Alteration of temperature is one of cancer therapies. In general, severe hyperthermia(around $43^{\circ}C$) and hypothermia(around $18^{\circ}C$) trigger apoptosis through mitochondria, though the specific mechanism is still unknown. CC-t6 and GB-d1 cell lines, which were originally derived from human cholangiocarcinoma and gall bladder cancer, were established from a metastatic lymph node. To investigate the mechanism of metastatic cancer cell response to thermal stresses, hyperthermia($37^{\circ}C{\rightarrow}43^{\circ}C$) and hypothermia($37^{\circ}C{\rightarrow}17.4^{\circ}C$) were designed. Thermal stresses did not induce apoptosis but necrotic cell death. Any alterations of caspase-3, -9, cytochrome c, Bax, and Bcl-2 were not found in both hyperthermia and hypothermia exposed fells using western blot analysis. In the transmission electron microscopy, typical necrotic, but not apoptotic, changes were observed. These results suggest that temperature changes induce cell death through necrotic pathway in metastatic cancer in vitro, and it can be one of effective anticancer methods.

암세포가 있는 장소의 온도를 변화시키는 것은 하나의 암 치료 방법이 될 수 있다. 명확한 기전은 아직 잘 밝혀져 있지 않지만, 고온은 미토콘드리아로 신호를 전달해서 cytochrome c를 분비시키는 세포자멸사로의 길로 유도하는 것으로 알려져 있다. 저온은 $30^{\circ}C$ 미만에서 세포자멸사를 유도하지만 심하지 않은 저온에서는($35{\sim}33^{\circ}C$ 혹은 $31{\sim}29^{\circ}C$)오히려 세포자멸사를 막는 것으로 알려져 있다. CC-t6와 GB-d1세포 주는 림프절로 전이된 사람의 담관암과 담낭암에서 확립한 것으로, 이와 같은 전이성 암세포가 온도 변화에 어떻게 반응을 하는지를 연구하기 위해 고온노출($37{\rightarrow}43^{\circ}C$)과 저온노출($37{\rightarrow}17.4^{\circ}C$)을 시행하였다. 세포의 종류나 온도 변화를 통한 스트레스의 방법과 관계없이 죽는 세포가 관찰되었으며, 고온노출이 가장 심한 영향을 주었다. 이런 죽어가는 세포는 세포자멸사가 아닌 세포괴사의 경로를 거치고 있었다. 투과전자현미경을 이용한 관찰에서 세포자멸사적인 모습은 보이지 않았고, caspase-3, -9, cytochrome c, Bax 같은 세포자멸사와 관련된 단백질의 변화도 관찰되지 않았고, 열충격단백질 70과 27도 증가하였다. 결국 CC-t6와 GB-d1 세포는 온도변화를 통한 스트레스를 주었을 경우 세포괴사로 죽음을 알 수 있었다. 온도변화를 통한 스트레스는 열충격단백질의 증가와 함께 세포괴사를 일으켰다. GB-d1과 CC-t6 세포에서 고온은 가장 심각하게 세포괴사를 일으켰으며, 저온은 초기에는 세포괴사를 유발하였으나 12시간 경과후에는 세포분열이 더욱 활발하게 일어나 세포의 생명력을 연장시켜주었다. 결국 이 실험에서는 전이성 암세포를 제거하는 방법으로는 고은이 가장 효과적이며 유용함을 알 수 있었다.

Keywords

References

  1. Arends MJ, Wyllie AH: Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32 : 223-254, 1991
  2. Agoff SN, Hou J, Linzer DI, Wu B: Regulation of the human hsp70 promoter by p53. Science 259 : 84-87, 1993 https://doi.org/10.1126/science.8418500
  3. AI-Fageeh MB, Marchant RJ, Carden MJ, Smales CM: The cold-shock response in cultured mammalian cells: harnessing the response for the improvement of recombinant protein production. Biotechnol Bioeng 93 : 829-835, 2006 https://doi.org/10.1002/bit.20789
  4. Aslam AF, Aslam AK, Vasavada BC, Khan IA: Hypothermia: evaluation, electrocardiographic manifestations, and management. Am J Med 119 : 297-301, 2006 https://doi.org/10.1016/j.amjmed.2005.09.062
  5. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK: Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol 12: 1539-1546, 2000 https://doi.org/10.1093/intimm/12.11.1539
  6. Benzeno S, Diehl JA: A novel WISp39 protein links Hsp90 and p21 stability to the G2/M checkpoint. Cancer Biol Ther 4 : 376-378, 2005 https://doi.org/10.4161/cbt.4.4.1700
  7. Binder RJ, Han DK, Srivastava PK: CD91: a receptor for heat shock protein gp96. Nat Immunol 1 : 151-155, 2000 https://doi.org/10.1038/77835
  8. Borst P, Rottenberg S: Cancer cell death by programmed necrosis? Drug Resist Updat 7: 321-324, 2004 https://doi.org/10.1016/j.drup.2004.11.003
  9. Buckley BA, Owen ME, Hofmann GE: Adjusting the thermostat: the threshold induction temperature for the heatshock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J Exp Biol 204 : 3571-3579, 2001
  10. Bursch W: The autophagosomal-Iysosomal compartment in programmed cell death. Cell Death Differ 8 : 569-581, 2001 https://doi.org/10.1038/sj.cdd.4400852
  11. Calderwood SK, Khaleque MA, Sawyer DB, Ciocca DR: Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci 31 : 164-172, 2006 https://doi.org/10.1016/j.tibs.2006.01.006
  12. Calderwood SK, Theriault JR, Gong J: Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity. Eur J Immunol 35 : 2518-2527, 2005 https://doi.org/10.1002/eji.200535002
  13. Calderwood SK: Chaperones and slow death-a recipe for tumor immunotherapy. Trends Biotechnol 23 : 57-59, 2005 https://doi.org/10.1016/j.tibtech.2004.12.011
  14. Christians ES, Zhou Q, Renard J, Benjamin IJ: Heat shock proteins in mammalian development. Semin Cell Dev Biol 14: 283-290, 2003 https://doi.org/10.1016/j.semcdb.2003.09.021
  15. Ciocca DR, Calderwood SK: Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones 10 : 86-103, 2005 https://doi.org/10.1379/CSC-99r.1
  16. Debatin KM: Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother 53: 153-159, 2004 https://doi.org/10.1007/s00262-003-0474-8
  17. Edwards MJ, Shiota K, Smith MS, Walsh DA: Hyperthermia and birth defects. Reprod Toxicol 9 : 411-425, 1995 https://doi.org/10.1016/0890-6238(95)00043-A
  18. Edwards MJ, Saunders RD, Shiota K: Effects of heat on embryos and foetuses. Int J Hyperthermia 19 : 295-324, 2003 https://doi.org/10.1080/0265673021000039628
  19. Ellis RJ, van der Vies SM: Molecular chaperones. Annu Rev Biochem 60: 321-347, 1991 https://doi.org/10.1146/annurev.bi.60.070191.001541
  20. Fairfield DA, Kanicki AC, Lomax MI, Altschuler RA: Induction of heat shock protein 32 (Hsp32) in the rat cochlea following hyperthermia. Hear Res 188 : 1-11, 2004 https://doi.org/10.1016/S0378-5955(03)00369-1
  21. Ghioni P, Bolognese F, Duijf PH, Van Bokhoven H, Mantovani R, Guerrini L: Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains. Mol Cell Biol 22 : 8659-8668, 2002 https://doi.org/10.1128/MCB.22.24.8659-8668.2002
  22. Hildebrandt B, Wust P, Ahlers O, Dieing A, Sreenivasa G, Kerner T, et al.: The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol 43 : 33-56, 2002 https://doi.org/10.1016/S1040-8428(01)00179-2
  23. Hornberg IJ, Bruggeman FJ, Westerhoff HV, Lankelma J: Cancer: a Systems Biology disease. Biosystems 83: 81-90, 2006 https://doi.org/10.1016/j.biosystems.2005.05.014
  24. Houshmand P, Zlotnik A: Targeting tumor cells. Current Opinion in Cell Biology 15 : 640-644, 2003 https://doi.org/10.1016/S0955-0674(03)00106-6
  25. Ito A, Honda H, Kobayashi T: Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of 'heat-controlled necrosis' with heat shock protein expression. Cancer Immunol Immunother 55 : 320-328, 2006 https://doi.org/10.1007/s00262-005-0049-y
  26. Jascur T, Brickner H, Salles-Passador I, Barbier V, El Khissiin A, Smith B, et al.: Regulation of p21 (WAF1/ CIP1) stability by WISp39, a Hsp90 binding TPR protein. Mol Cell 17 : 237-249, 2005 https://doi.org/10.1016/j.molcel.2004.11.049
  27. Kaufmann SH, Earnshaw WC: Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256 : 42-49, 2000 https://doi.org/10.1006/excr.2000.4838
  28. Kaufmann SH, Earnshaw WC: Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256: 42-49, 2000 https://doi.org/10.1006/excr.2000.4838
  29. Leist M, Jaattela M: Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2 : 589-598, 2001 https://doi.org/10.1038/35085008
  30. Lepock JR: Cellular effects of hyperthermia: relevance to the minimum dose for thermal damage. Int J Hyperthermia 19 : 252-266, 2003 https://doi.org/10.1080/0265673031000065042
  31. Lindquist S, Craig EA: The heat-shock proteins. Annu Rev Genet 22 : 631-677, 1998 https://doi.org/10.1146/annurev.ge.22.120188.003215
  32. Liu G, Lozano G: p21 stability: linking chaperones to a cell cycle checkpoint. Cancer Cell 7 : 113-114, 2005 https://doi.org/10.1016/j.ccr.2005.01.019
  33. Madden SL, Galella EA, Zhu J, Bertelsen AH, Beaudry GA: SAGE transcript profiles for p53-dependent growth regulation. Oncogene 15 : 1079-1085, 1997 https://doi.org/10.1038/sj.onc.1201091
  34. Masse D, Ebstein F, Bougras G, Harb J, Meflah K, Gregoire M: Increased expression of inducible HSP70 in apoptotic cells is correlated with their efficacy for antitumor vaccine therapy. Int J Cancer 111 : 575-583, 2004 https://doi.org/10.1002/ijc.20249
  35. Mesner PW Jr, Budihardjo II, Kaufmann SH: Chemotherapy-induced apoptosis. Adv Pharmacol 41 : 461-499, 1997 https://doi.org/10.1016/S1054-3589(08)61069-8
  36. Mirkes PE: 2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development. Teratology 65: 228-239, 2002 https://doi.org/10.1002/tera.10049
  37. Nylandsted J, Brand K, Jaattela M: Heat shock protein 70 is required for the survival of cancer cells. Ann NY Acad Sci 926: 122-125, 2000 https://doi.org/10.1111/j.1749-6632.2000.tb05605.x
  38. Proskuryakov SY, Konoplyannikov AG, Gabai VL: Necrosis: a specific form of programmed cell death? Exp Cell Res 283: 1-16, 2003 https://doi.org/10.1016/S0014-4827(02)00027-7
  39. Russotti G, Brieva TA, Toner M, Yarmush ML: Induction of tolerance to hypothermia by previous heat shock using human fibroblasts in culture. Cryobiology 33 : 567-580, 1996 https://doi.org/10.1006/cryo.1996.0060
  40. Shibano T, Morimoto Y, Kemmotsu O, Shikama H, Hisano K, Hua Y: Effects of mild and moderate hypothermia on apoptosis in neuronal PC12 cells. Br J Anaesth 89 : 301-305, 2002 https://doi.org/10.1093/bja/aef181
  41. Srivastava PK, Udono H, Blachere NE, Li Z: Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 39 : 93-98, 1994
  42. Srivastava PK: Hypothesis: controlled necrosis as a tool for immunotherapy of human cancer. Cancer Immun 3 : 4, 2003
  43. Thayer JM, Mirkes PE: Induction of Hsp72 and transient nuclear localization of Hsp73 and Hsp72 correlate with the acquisition and loss of thermotolerance in postimplantation rat embryos. Dev Dyn 208: 227-243, 1997 https://doi.org/10.1002/(SICI)1097-0177(199702)208:2<227::AID-AJA10>3.0.CO;2-0
  44. Theodorakis NG, Drujan D, De Maio A: Thermotolerant cells show an attenuated expression of Hsp70 after heat shock. J Biol Chem 274: 12081-12086, 1999 https://doi.org/10.1074/jbc.274.17.12081
  45. Tsutsumi-Ishii Y, Tadokoro K, Hanaoka F, Tsuchida N: Response of heat shock element within the human HSP70 promoter to mutated p53 genes. Cell Growth Differ 6 : 1-8, 1995
  46. van der Zee J: Heating the patient: a promising approach? Ann Oncol 13 : 1173-1184, 2002 https://doi.org/10.1093/annonc/mdf280
  47. Yuan J, Lipinski M, Degterev A: Diversity in the mechanisms of neuronal cell death. Neuron 40: 401-413, 2003 https://doi.org/10.1016/S0896-6273(03)00601-9
  48. Zong WX, Ditsworth D, Bauer DE, Wang ZQ, Thompson CB: Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev 18 : 1272-1282, 2004 https://doi.org/10.1101/gad.1199904