Journal of Oral Medicine and Pain

Korean Academy of Orofacial Pain and Oral Medicine (대한안면통증구강내과학회)
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Mechanism Underlying a Proteasome Inhibitor, Lactacystin-Induced Apoptosis on SCC25 Human Tongue Squamous Cell Carcinoma Cells

사람혀편평상피세포암종세포에서 proteasome 억제제인 lactacystin에 의해 유도된 세포자멸사의 기전에 대한 연구

  • Baek, Chul-Jung (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Kim, Gyoo-Cheon (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Kim, In-Ryoung (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Lee, Seung-Eun (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Kwak, Hyun-Ho (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Park, Bong-Soo (Dept. of Oral Anatomy, School of Dentistry, Pusan National University) ;
  • Tae, Il-Ho (Dept. of Oral Medicine, School of Dentistry, Pusan National University) ;
  • Ko, Myung-Yun (Dept. of Oral Medicine, School of Dentistry, Pusan National University) ;
  • Ahn, Yong-Woo (Dept. of Oral Medicine, School of Dentistry, Pusan National University)
  • 백철중 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 김규천 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 김인령 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 이승은 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 곽현호 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 박봉수 (부산대학교 치의학전문대학원 구강해부학교실) ;
  • 태일호 (부산대학교 치의학전문대학원 구강내과학교실) ;
  • 고명연 (부산대학교 치의학전문대학원 구강내과학교실) ;
  • 안용우 (부산대학교 치의학전문대학원 구강내과학교실)
  • Published : 2009.09.30
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Abstract

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

Sreptomyces라는 세균에서 추출한 lactacystin은 선택적인 proteasome 억제제로서 많은 연구에서 사용되어져 왔다. Proteasome 억제제는 최근의 많은 연구를 통해서 암세포증식의 억제에 대한 효과가 증명되었으며, 특히 다른 항암제와 병용처리 시, 상호작용에 의한 상승효과가 있다고 알려져 있다. 현재 proteasome 억제제는 새로운 강력한 항암제로서 분류되어 있다. 본 연구는 사람혀편평세포암종세포(SCC25 cells)에서 lactacystin의 세포독성과 성장억제 효과, 그리고 세포자멸사의 유도에 대한 분자생물학적 기전을 밝히기 위해 실험을 시행하였다. SCC25 세포, 사람정상각화세포 (HaCaT cells) 그리고 사람치은섬유모세포(HGF-1 cells)의 생존율 측정은 MTT법을 시행하였고, SCC25 세포의 성장억제를 확인하기 위해서는 clonogenic assay를 사용하였다. lactcystin이 SCC25 세포에서 세포자멸사가 유도되는지를 확인하기 위해서 hoechst 염색법, hemacolor 염색법 그리고 TUNEL법을 시행하였다. 그리고 SCC25 세포에 lactacystin을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이저주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하였다. Lactacystin으로 처리된 SCC25 세포는 시간 및 용량 의존적인 세포생존율의 감소, 용량의존적인 세포성장억제 그리고 세포자멸사에 의한 세포죽음을 보였다. 흥미롭게도 lactacytin은 정상세포인 HaCat 세포와 HGF-1 세포에서는 세포독성을 전혀 보이지 않았다. 그리고 lactacystin이 적용된 SCC25세포에서 핵 응축, DNA의 조각남, 사립체막전위와 proteasome 활성도의 감소, DNA 양의 감소, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, Bax의 증가, caspase-7, caspase-3, PARP, lamin A/C 그리고 DFF45 (ICAD)의 활성화 혹은 파괴와 같은 아주 다양한 세포자멸사 증거를 보였다. Flow cytometry 분석에서는 CDK 억제제인 $p21^{WAF1/CIP1}$와 $p27^{KIP1}$의 발현 증가와 관계있는 것으로 추정되어 지는 G1 세포주기 정지를 보였다. 이러한 결과는 lactacytin이 SCC25 세포에서 G1 세포주기정지와 proteasome, 사립체 및 caspase 경로의 연속반응을 통한 세포자멸사를 유도함을 명확하게 증명하고 있다. 이와 같은 세포주기 정지와 세포자멸사 유도능은 lactacytin이 사람혀편평상피세포암종의 새로운 치료전략으로서의 가능성을 제공한다고 생각한다.

Keywords

References

  1. Carson DA, Ribeiro JM. Apoptosis and disease. Lancet 1993;341:1251-1254 https://doi.org/10.1016/0140-6736(93)91154-E
  2. Ohta K, Yamashita N. Apoptosis of eosinophils and lymphocytes in allergic inflammation. J Allergy Clin Immunol 1999;104:14-21 https://doi.org/10.1016/S0091-6749(99)70107-7
  3. Williams GT. Programmed cell death: apoptosis and oncogenesis. Cell 1991;65:1097-1098 https://doi.org/10.1016/0092-8674(91)90002-G
  4. Thornberry NA, Rosen A, Nicholson DW. Control of apoptosis by proteases. Adv In Pharmacol 1997;41: 155-177 https://doi.org/10.1016/S1054-3589(08)61058-3
  5. Green DR and Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-1312 https://doi.org/10.1126/science.281.5381.1309
  6. Orlowski RZ. The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ 1999;6:303-313 https://doi.org/10.1038/sj.cdd.4400505
  7. Drexler HC. Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci USA 1997;94:855-860 https://doi.org/10.1073/pnas.94.3.855
  8. Orlowski RZ, Eswara JR, Lafond-Walker A, Grever MR, Orlowski M, Dang CV. Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor. Cancer Res 1998;58:4342-4348
  9. Meng L, Kwok BH, Sin N, Crews CM. Eponemycin exerts its antitumor effect through the inhibition of proteasome function. Cancer Res 1999;59:2798-2801
  10. Drexler HC, Risau W, Konerding MA. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J 2000;14:65-77 https://doi.org/10.1096/fasebj.14.1.65
  11. Delic J, Masdehors P, Omura S et al. The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo- and radio-resistant human chronic lymphocytic leukemia lymphocytes to TNF-alphainitiated apoptosis. Br J Cancer 1998;77:1103-1107 https://doi.org/10.1038/bjc.1998.183
  12. Lin ZP, Boller YC, Amer SM et al. Prevention of brefeldin A- induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance. Cancer Res 1998;58:3059-3069
  13. Adams J. Development of the proteasome inhibitor PS-341. Oncologist 2002;7:9-16
  14. An WG, Hwang SG, Trepel JB, Blagosklonny MV. Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition. Leukemia 2000;14:1276-1283 https://doi.org/10.1038/sj.leu.2401812
  15. Pasquini LA, Besio Moreno M, Adamo AM, Pasquini JM, Soto EF. Lactacystin, a specific inhibitor of the proteasome, induces apoptosis and activates caspase-3 in cultured cerebellar granule cells. J Neurosci Res. Mar 2000;59:601-611 https://doi.org/10.1002/(SICI)1097-4547(20000301)59:5<601::AID-JNR3>3.0.CO;2-1
  16. Inoue T, Shiraki K, Fuke H et al. Proteasome inhibition sensitizes hepatocellular carcinoma cells to TRAIL by suppressing caspase inhibitors and AKT pathway. Anticancer Drugs. 2006;17:261-268 https://doi.org/10.1097/00001813-200603000-00004
  17. Kudo Y, Takata T, Ogawa I et al. p27Kip1 accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. Clin Cancer Res 2000;6:916-923
  18. Takezawa J, Ishimi Y, Yamada K. Proteasome inhibitors remarkably prevent translesion replication in cancer cells but not normal cells. Cancer Sci 2008;99: 863-871 https://doi.org/10.1111/j.1349-7006.2008.00764.x
  19. Shen J, Huang C, Jiang L et al. Enhancement of cisplatin induced apoptosis by suberoylanilide hydroxamic acid in human oral squamous cell carcinoma cell lines. Biochem Pharmacol 2007;73: 1901-1909 https://doi.org/10.1016/j.bcp.2007.03.009
  20. Adams J, Palombella VJ, Sausville EA et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999;59: 2615-2622
  21. Kim JH, Bae HR, Park BS et al. Early mitochondrial hyperpolarization and intracellular alkalinization in lactacystin-induced apoptosis of retinal pigment epithelial cells. J Pharmacol Exp Ther 2003;305: 474-481 https://doi.org/10.1124/jpet.102.047811
  22. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68: 251-306 https://doi.org/10.1016/S0074-7696(08)62312-8
  23. Grimm LM, Goldberg AL, Poirier GG, Schwartz LM, Osborne BA. Proteasome play an essential role in thymocyte apoptosis. EMBO J 1996;15:3845-3852
  24. Li B, Dou QP. Bax degradation by the ubiquitin/ proteasome-dependent pathway: Involvement in tumor survival and progression. Pro Natl Acad Sci USA 2000;97:3850-3855 https://doi.org/10.1073/pnas.070047997
  25. 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
  26. Susin SA, Lorenzo HK, Zamzami N et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441-446 https://doi.org/10.1038/17135
  27. Orrenius S. Mitochondrial regulation of apoptotic cell death. Toxicol Lett 2004;149:19-23 https://doi.org/10.1016/j.toxlet.2003.12.017
  28. Barczyk K, Kreuter M, Pryjma J et al. Serum cytochrome c indicates in vivo-apoptosis and it can serve as a prognostic marker during cancer therapy. In. J Cancer 2005;114:167–173
  29. Brouckaert G, Kalai M, Saelens X, Vandenabeele P. Apoptotic Pathways and Their Regulation. In: Los, M., Gibson, S.B. (Eds.), Apoptotic Pathways as Target for Novel Therapies in Cancer and Other Diseases. New York. 2005, Springer Academic Press
  30. Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770-776 https://doi.org/10.1038/35037710
  31. Hunter JJ, Parslow TG. A peptide sequence from Bax that converts Bcl-2 into an activator of apoptosis. J Biol Chem 1996;271:8521-8524 https://doi.org/10.1074/jbc.271.15.8521
  32. Narita M, Shimizu S, Ito T et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria.Proc Natl Acad Sci U S A. 1998;95:14681-14686 https://doi.org/10.1073/pnas.95.25.14681
  33. Wagenknecht B, Hermission M, Groscurth P, Liston P, Krammer PH, Weller M. Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release. J Neurochem 2000;75:2288-2297 https://doi.org/10.1046/j.1471-4159.2000.0752288.x
  34. Marshansky V, Wang X, Bertrand R et al. Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells. J Immunol 2001;166:3130-3142 https://doi.org/10.4049/jimmunol.166.5.3130
  35. Li P, Nijhawan D, Budihardjo I et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479-489 https://doi.org/10.1016/S0092-8674(00)80434-1
  36. Zou H, Li Y, Liu X, Wang, X. An APAF-1, cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999;274:11549–11556 https://doi.org/10.1074/jbc.274.17.11549
  37. Dauglas E, Susin SA, Zamzami N et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000;14:729-739 https://doi.org/10.1096/fasebj.14.5.729
  38. Thornberry NA, Lazebnik Y. Caspase: Enemies within. Science 1998;28:1312-1316
  39. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. Three-dimensional structure of the apoptosome: Implications for assembly, procaspase-9 binding, and activation. Mol Cell 2002;9:423-432 https://doi.org/10.1016/S1097-2765(02)00442-2
  40. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899-1911 https://doi.org/10.1101/gad.13.15.1899
  41. Porter AG. Protein translocation in apoptosis. Trends Cell Biol 1999;9:394-401 https://doi.org/10.1016/S0962-8924(99)01624-4
  42. Cheng AC, Jian CB, Huang YT, Lai CS, Hsu PC, Pan MH. Induction of apoptosis by Uncaria tomentosa through reactive oxygen species production, cytochrome c release, and caspases activation in human leukemia cells. Food Chem Toxicol 2007;45:2206-2218 https://doi.org/10.1016/j.fct.2007.05.016
  43. Pavletich NP. Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol. 1999;287: 821-828 https://doi.org/10.1006/jmbi.1999.2640
  44. Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL. Inhibition of proteasome activities and subunit specific-amino terminal threonine modification by lactocystin. Science 1995;268:726–731 https://doi.org/10.1126/science.7732382
  45. Shinohara K, Tomioka M, Nakano H, Tone S, Ito H, Kawashima S. Apoptosis induction resulting from proteasome inhibition. Biochem J 1996;317:385-388 https://doi.org/10.1042/bj3170385
  46. Meriin AB, Gabai VL, Yaglom J, Shifrin VI, Sherman MY. Proteasome inhibitors activate stress kinases and induce Hsp72. Diverse effects on apoptosis. J Biol Chem 1998;273:6373-6379 https://doi.org/10.1074/jbc.273.11.6373
  47. Elledge S, Harper W. Cdk inhibitors: on the threshold of checkpoints and development. Curr Op Cell Biol 1994;6:847–852 https://doi.org/10.1016/0955-0674(94)90055-8
  48. Pagano M, Tam SW, Theodoras AM et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 1995;269:682–685 https://doi.org/10.1126/science.7624798