嗜伊紅血球在人類免疫系統中扮演很重要的角色，當嗜伊紅血球被激發時，許多的細胞激素與具有細胞毒性的嗜伊紅血球陽離子蛋白(eosinophil cationic protein, ECP)將被釋放出來。ECP是一種具有低活性的核醣核酸酶，目前已經被廣泛的使用在氣喘疾病的偵測上，此蛋白是一個很好的氣喘指標。ECP已知會抑制細胞生長，然而它所引起的細胞死亡的機制仍尚未被討論過。在本實驗中有了新的發現，我們發現重組ECP(recombinant ECP, rECP)會導致細胞凋零並且是透過Caspase-8的途徑。首先，我們先確認rECP造成人類支氣管表皮細胞(Beas-2B)的影響，並且測出抑制一半生長的濃度(IC50)為21.03 uM. 我們利用檢視細胞核的萎縮、PARP的裂解、Sub G1與Annexin V來判斷此細胞是經由細胞凋零而死亡。為了找出是透過哪一種細胞凋零途徑，Caspase的廣泛抑制劑(Z-VAD-FMK)與Caspase-8的抑制劑(Z-IETD-FMK)將用來證明其的路徑，更進一步的發現顯示當細胞在rECP 的處理之下，Caspase-8會裂解而被活化。雖然細胞經歷細胞凋零，但是粒線體與內質網所引起的細胞凋零途徑似乎沒有明顯的參予於其中。我們亦發現了當細胞在rECP處理下，會釋放出細胞素－腫瘤壞死因子(TNF-a□於培養液中，因此我們推測Caspase-8 所參予的細胞凋零途徑是被功能如同自主免疫的TNF-□所激發。我們同時也推測各種核醣核酸酶如ECP、EDN與RNase A之細胞毒性是依據核醣核酸本生的等位點而不同。最後，我們發現rECP亦會引起肺表皮癌細胞(A549)產生細胞凋零，此發現將對癌細胞的治療方針上有幫助。

The purpose of this study is to investigate the effects of eosinophil cationic protein (ECP) on human bronchial epithelial cells (Beas-2B cells). Eosinophilic granulocytes are important for immune system in human body. Many derived cationic proteins with cytotoxic activities such as ECP and eosinophil derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, has been widely used to be a biomarker for asthma. ECP is reported to inhibit cell viability, but it is never fully verified its mechanism of cell death. In this study, it was the novel discovery that rECP could cause cell apoptosis and involve in caspase-8 pathway in Beas-2B cells. First, we found that rECP could inhibit the cell viability in Beas-2B cells with IC50 of 21.03 uM. The cell death undergoing apoptotic pathway was investigated by chromatin condensation, cleavage of PARP, sub-G1 of cell cycle and Annexin V. To elucidate the specific apoptotic pathway, the general caspase inhibitor (Z-VAD-FMK) and caspase-8 inhibitor (Z-IETD-FMK) were used to prove the caspase and caspase-8 dependent apoptosis. Further, the cleavage of caspase 8 was also detected after rECP treatment. Although apoptosis occurred, both mitochondrial membrane potential (MMP) and endoplasmic reticulum (ER) response were not obviously involved in the rECP-induced apoptosis. We also found that TNF-□ was released from Beas-2B cells into medium during rECP-induced apoptosis. Therefore, we hypothesized that caspase-8 mediated the specific apoptosis pathway which was trigger by TNF-a functioned as autocrine. We also assumed that the cytotoxicites of different RNases such as ECP, EDN and RNase A depended on their isoelectric pH (pI). Finally, we found that rECP also induced apoptosis in lung carcinoma cells (A549 cells).

1. Rothenberg, M. E., and Hogan, S. P. (2006) Annu. Rev. Immunol. 24, 147-174
2. Stenfeldt, A. L., and Wenneras, C. (2004) Immunology 112(4), 605-614
3. Venge, P., Bystrom, J., Carlson, M., Hakansson, L., Karawacjzyk, M., Peterson, C., Seveus, L., and Trulson, A. (1999) Clinical and Experimental Allergy 29(9), 1172-1186
4. Durham, S. R., Loegering, D. A., Dunnette, S., Gleich, G. J., and Kay, A. B. (1989) Journal of Allergy and Clinical Immunology 84(6), 931-936
5. Walsh, G. M. (2001) Current Opinion in Hematology 8(1), 28-33
6. Boix, E., Leonidas, D. D., Nikolovski, Z., Nogues, M. V., Cuchillo, C. M., and Acharya, K. R. (1999) Biochemistry 38(51), 16794-16801
7. Motojima, S., Frigas, E., Loegering, D. A., and Gleich, G. J. (1989) Am. Rev. Respir. Dis. 139(3), 801-805
8. Carreras, E., Boix, E., Navarro, S., Rosenberg, H. F., Cuchillo, C. M., and Nogues, M. V. (2005) Molecular and Cellular Biochemistry 272(1-2), 1-7
9. Ali, S., Kaur, J., and Patel, K. D. (2000) American Journal of Pathology 157(1), 313-321
10. Venge, P. (2004) Allergy 59(1), 26-32
11. Rosenberg, H. F. (1995) Journal of Biological Chemistry 270(14), 7876-7881
12. Fan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y., and Chang, M. D. (2007) Traffic 8(12), 1778-1795
13. Koh, G. C. H., Shek, L. P. C., Goh, D. Y. T., Van Bever, H., and Koh, D. S. Q. (2007) Respiratory Medicine 101(4), 696-705
14. Vila, L., Sanz, M. L., Sanchez-Lopez, G., Garcia-Aviles, G., and Dieguez, I. (2001) Allergy 56(6), 568-572
15. Wu, C. M., and Chang, M. D. T. (2004) Biochemical and biophysical research communications 322(2), 585-592
16. Chang, H. T., Kao, Y. L., Wu, C. M., Fan, T. C., Lai, Y. K., Huang, K. L., Chang, Y. S., Tsai, J. J., and Chang, M. D. T. (2007) Journal of Cellular Biochemistry 100(5), 1266-1275
17. Trautmann, A., Schmid-Grendelmeier, P., Kruger, K., Crameri, R., Akdis, M., Akkaya, A., Brocker, E. B., Blaser, K., and Akdis, C. A. (2002) Journal of Allergy and Clinical Immunology 109(2), 329-337
18. Dricu, A., Sergiu-Bogdan, C., Brismar, K., Biberfeld, P., and Andersson, L. C. (2004) Anticancer Research 24(3A), 1427-1432
19. Tesfaigzi, Y. (2006) American Journal of Respiratory Cell and Molecular Biology 34(5), 537-547
20. Jyonouchi, H. (1999) Apoptosis 4(6), 407-417
21. Amstad, P., Reddel, R. R., Pfeifer, A., Malanshibley, L., Mark, G. E., and Harris, C. C. (1988) Molecular Carcinogenesis 1(3), 151-160
22. Kinnula, V. L., Raivio, K. O., Linnainmaa, K., Ekman, A., and Klockars, M. (1995) Free Radical Biology and Medicine 18(3), 391-399
23. Pichavant, M., Charbonnier, A. S., Taront, S., Brichet, A. B., Wallaert, B., Pestel, J., Tonnel, A. B., and Gosset, P. (2005) Journal of Allergy and Clinical Immunology 115(4), 771-778
24. Fesik, S. W., and Shi, Y. G. (2001) Science 294(5546), 1477-1478
25. Kroemer, G., and Martin, S. J. (2005) Nature Medicine 11(7), 725-730
26. Earnshaw, W. C. (1999) Nature 397(6718), 387-+
27. Philchenkov, A. (2004) Journal of Cellular and Molecular Medicine 8(4), 432-444
28. Riedl, S. J., and Shi, Y. G. (2004) Nature Reviews Molecular Cell Biology 5(11), 897-907
29. Ferri, K. F., and Kroemer, G. (2001) Nature Cell Biology 3(11), E255-E263
30. Kroemer, G., Galluzzi, L., and Brenner, C. (2007) Physiological Reviews 87(1), 99-163
31. Nakanishi, K., Dohmae, N., and Morishima, N. (2007) Faseb Journal 21(11), 2994-3003
32. Yu, Z. F., Luo, H., Fu, W. M., and Mattson, M. P. (1999) Experimental Neurology 155(2), 302-314
33. Kim, P. K. M., Park, S. Y., Koty, P. P., Hua, Y., Luketich, J. D., and Billiar, T. R. (2003) Journal of Thoracic and Cardiovascular Surgery 125(6), 1336-1342
34. Krieghoff, E., Milovic-Holm, K., and Hofmann, T. G. (2007) Cell Cycle 6(7), 771-775
35. Lisa, R. J., Borysenko, C. W., and Blair, H. (2007) Ann N Y Acad Sci
36. Shankar, S., Siddiqui, I., and Srivastava, R. K. (2007) Molecular and Cellular Biochemistry 304(1-2), 273-285
37. Aktas, O., Schulze-Topphoff, U., and Zipp, F. (2007) Frontiers in Bioscience 12, 2912-2921
38. Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., and Bairoch, A. (2003) Nucleic Acids Research 31(13), 3784-3788
39. Munthe-Kaas, M. C., Gerritsen, J., Carlsen, K. H., Undlien, D., Egeland, T., Skinningsrud, B., Torres, T., and Carlsen, K. L. (2007) Allergy 62(4), 429-436
40. Kim, C. K., Kim, S. W., Kim, Y. K., Kang, H., Yu, J. H., Yoo, Y., and Koh, Y. Y. (2005) Clinical and Experimental Allergy 35(5), 591-597
41. Kim, C. K., Kim, J. T., Kang, H., Yoo, Y., and Koh, Y. Y. (2003) Clinical and Experimental Allergy 33(10), 1409-1414
42. Boix, E., Nikolovski, Z., Moiseyev, G. P., Rosenberg, H. F., Cuchillo, C. M., and Nogues, M. V. (1999) Journal of Biological Chemistry 274(22), 15605-15614
43. Hansen, M. B., Nielsen, S. E., and Berg, K. (1989) Journal of Immunological Methods 119(2), 203-210
44. Partheniou, F., Kelsey, S. M., Srinivasula, S. M., Newland, A. C., Alnemri, E. S., and Jia, L. (2001) Biochemical and biophysical research communications 287(1), 181-189
45. Chang, Y. S., Lo, C. W., Sun, F. C., Chang, M. D., and Lai, Y. K. (2006) Biochemical and biophysical research communications 344(1), 37-44
46. Ibuki, Y., and Goto, R. (2000) Biochemical and biophysical research communications 279(3), 872-878
47. Reddy, R. K., Mao, C., Baumeister, P., Austin, R. C., Kaufman, R. J., and Lee, A. S. (2003) The Journal of biological chemistry 278(23), 20915-20924
48. Debatin, K. M., Poncet, D., and Kroemer, G. (2002) Oncogene 21(57), 8786-8803
49. Iordanov, M. S., Ryabinina, O. P., Wong, J., Dinh, T. H., Newton, D. L., Rybak, S. M., and Magun, B. E. (2000) Cancer Research 60(7), 1983-1994
50. Hu, C. C., Tang, C. H., and Wang, J. J. (2001) FEBS Lett 503(1), 65-68
51. Newton, D. L., and Rybak, S. M. (1998) Journal of the National Cancer Institute 90(23), 1787-1791
52. Masud, A., Mohapatra, A., Lakhani, S. A., Ferrandino, A., Hakem, R., and Flavell, R. A. (2007) Journal of Biological Chemistry 282(19), 14132-14139
53. Polito, A. J., and Proud, D. (1998) Journal of Allergy and Clinical Immunology 102(5), 714-718
54. Takizawa, H., Ohtoshi, T., Kawasaki, S., Abe, S., Sugawara, I., Nakahara, K., Matsushima, K., and Kudoh, S. (2000) Respirology (Carlton, Vic 5(2), 197-203
55. Pimentel-Muinos, F. X., and Seed, B. (1999) Immunity 11(6), 783-793
56. Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., Ackerman, S. J., and McKean, D. J. (1986) Proceedings of the National Academy of Sciences of the United States of America 83(10), 3146-3150
57. Berry, L., Andrew, M., Post, M., Ofosu, F., and O'Brodovich, H. (1991) Am J Respir Cell Mol Biol 4(4), 338-346
58. Fuchs, S. M., and Raines, R. T. (2005) Protein Sci 14(6), 1538-1544