摘要
抗菌肽是一类带正电、多具双亲性的小分子多肽,由于其独
特的结构,抗菌肽对多种细菌和癌细胞有强烈的抑制作用,且多
数抗菌肽在发挥以上作用时对正常细胞没有破坏作用。
Tachyplesin-I是鲎(Tachypleus tridentatus)血液中的一类具β结
构的环状抗菌肽,抗菌作用强烈,是进行抗癌机制研究的一个好
的模板。
本课题以人低分化胃腺癌BGC-823细胞株为模型,探讨人工
化学合成抗菌肽tachyplesin-I对癌细胞的生物学效应和可能的作
用机制。为此,我们首先测定了tachyplesin-I对多种肿瘤细胞存
活率的影响及对BGC-823细胞生长的抑制作用,观察了
tachyplesin-I作用下细胞的形态学变化;然后主要从对膜作用角
度入手,检测与观察荧光探针FDA、Dextran-FITC、JC-1、
YO-PRO-1的强度和分布,分析tachyplesin-I作用后细胞膜、细
胞核和线粒体膜系统的变化。
结果显示:BGC-823细胞的存活率及生长动态受到很大影响;
细胞形态也发生变化,细胞膜出现不完整甚至大面积破损崩溃现
象,线粒体肿胀,次级溶酶体增生,部分细胞核膜崩溃;荧光分
光光度计检测到tachyplesin-I 2小时处理后各组的相对荧光
强度增加,说明荧光均有外泄,细胞膜均受到损伤,低于50 μg/mL
的实验组相对荧光强度在15. 14-17. 84之间,而50 μg/ml以上的
实验组相对荧光强度在60. 54-67. 57之间,存在阈值作用;激光
扫描共聚焦显微镜下观察到Dextran-FITC经2小时处理未能进入
细胞内部,24小时处理后,细胞允许40KD的大分子探针进入,
说明经长时间作用细胞膜受到较大的损伤;正常人红细胞在
tachyplesin-I作用下发生溶血作用,溶血率在26. 66%-44. 39%
之间;激光扫描共聚焦显微镜观察到JC-1染色由强红色变弱,大
多数呈绿色,流式细胞仪检测到绿色荧光强度有所升高,红色荧
光强度大幅降低,说明线粒体膜电位去极化;流式细胞仪检测到
YO干 1绿色荧光增强,说明核膜通透性增加。
结论如下:人工合成抗菌肽tachaplesin刁通过细胞杀伤的途
径抑制肿瘤细胞;作用机制首先是通过对膜作用,其后tachyplesin-
I可能进一步通过对膜作用而使膜上孔洞不断扩大或迅速进入细
胞内部作用在核与线粒体上达到对肿瘤细胞的杀伤低浓度和高
浓度tachyplesin-I对膜有不同的作用方式,但只有高浓度的
tachyplesin刁最终造成细胞死亡。
Antimicrobial peptides, the majority of which are cationic and amphipathic, are small peptides. They can strongly act on many kinds of bacterial and cancer cells, while at the same concentrations they act weakly on normal cells. The ability is due to their unique structures. This selective toxicity has attracted much attention. Now some progress has made on the linear antimicrobial peptides in inhibiting bacterial and cancer cells but not in the case of cyclic peptides. Tachyplesin- I , a small antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus ) , which has a cyclic anti-parallel 3 -sheet structure held together by two disulfide bonds, permeabilizes both bacterial and artificial lipid membranes at low concentrations. It suggests that tachyplesin-1 is also a potent anticancer agent.
In this experiment, we aim at exploring whether, how and to what extent the synthetical antimicrobial peptide tachyplesin inhibits human gastric adenocarcinoma BGC-823 cell. Firstly, we test the effect of tachyplesin- I on cell proliferation, livability and morphology , and then we test the integrity of vital membranes via the change of intensity and distribution of FDA Dextran-FITC JC-1 and YO-PRO-1 probes.
The results show that tachyplesin-1 inhibited the proliferation of BGC-823 cell and reduced the survival ratio of cancer cells. Observation from transmission electron microscope shows tachyplesin- I broke cell membranes and nucleus membranes,
induced mitochondria swelling and increased the number of secondary lysosome. Staining with fluorescent probes of FDA Dextran-FITC JC-1 and YO-PRO-1 indicated that tachyplesin- I could permeabilize cell membranes, induce potential shift of mitochondria membrane and damage on nucleus membranes. Hemolytic test indicated tachyplesin- I could damage on human erymrocyte membranes at low level.
Based on what we have found, we can give a general idea about the anticancer mechanism of tachyplesin- I : 1 ) Synthetical antimicrobial peptide tachyplesin-1 inhibits the BGC-823 cell via the pass way of cytotoxicity; 2) Interaction of the peptide with membrane is the first step, and then possibly there are two ways, the big pores form, or the peptide enters the cell to reach an alternative target, including mitochondria and nucleus; 3 ) Tachyplesin- I interacts with membrane via two ways depending on its concentrations.
引文
1. Boman HG. Antibacterial peptides:Key components needed in immunity. Cell 1991,65:205-207
2. Lehrer RI, Lichtenstein Ak, Ganz T. Defensins: Antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol. 1993, 11:105-128
3. Selsted Me, Novotny Mj, Morris WL, et al. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem. 1992,267:4292-4295
4. Zanetti M, Litteri L, Gennaro R, Hostmann H, Romeo D. Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules. J Cell Biol. 1990,111:1363-1371
5. Hoffmann JA. Innate immunity of insects. Curr Opin Immunol. 1995, 7(1) : 4-10
6. Dimarcq JL. Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae . EMBO J. 1990, 9(8) : 2507-2515
7. Matsuzaki K. Why and how are peptide-lipid interations utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta. 1999, 1462: 1-10
8、 屈贤铭,唐海伦,Hakan S.柞蚕杀菌肽B、D对脂质体的作用. 生物化学与生物物理学报.1989,21(1) :35-41
9. Epand RM, Vogel HJ. Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta. 1999, 1462:11-28
10. Christensen B, Fink J, Merrifield RB, et al. Channel-forming properties of cecropins and related model compounds
incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA. 1988, 85(14) : 5072-5076
11. Sivestro L, Gupta K, Weiser JN, et al. The concentration-dependent membrane activity of Cecropin A. Biochemistry. 1997,36:11452-11460
12. Heller WT, Waring AJ, Lehrer RI, et al. Membrane thinning effect of the β-sheet antimicrobial protegrin. Biochemistry. 2000,39:139-145
13. Gazit E, Boman A, Boman HG et al. Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles. Biochemistry 1995, 34(36) :11479-11488
14. Wu M, Maier E, Benz R, Hancock RW. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry. 1999,38:7235-7242
15. Park CB, Kim HS and Kim SC. Mechanism of action of the antimicrobial peptide buforin Ⅱ: Buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem. Biophys. Res. Commun. 1998, 244(1) : 253-257
16. Kanyshkova TG, Semenov DV, Buneva VN, et al. Human milk lactoferrin binds two DNA molecules with different affinities. FEBS lett. 1999,451:235-237
17. Subbalakshmi C and Sitaram N. Mechanism of antimicrobial action of indolicidin. FEMS Microbiol. Lett. 1998,160(1) :91-96
18. Boman HG, Agerberth B and Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial
peptides from pig intestine. Infect. Immun. 1993,61:2978-2984
19. Chen HM, Wang W, Smith D, et al. Effects of the anti-bacterial peptide cecropin Band its analogs, cecropins B-1 and B-2, on liposomes, bacteria, and cancer cells. Biochim Biophys. Acta. 1997, 1336:171-179
20. Srisailam S, Arunkumar AI, Wang W, et al. Conformational study of a custom antibacterial peptide cecropin B1:implications of the lytic activity. Biochim Biophys. Acta. 2000, 1479:275-285
21. Jaynes JM, Julian GR, Jeffers GW, et al. Invitro cytocidal effect of lytic peptides on several transformed mammalian cell lines. Pept Res. 1989,2(2) : 157-160
22、 赵东红,张双全,戴祝英等.重组家蚕抗菌肽CM4对癌细胞 骨架及核骨架破坏作用的观察.高技术通讯.2001,(1) :23-27
23. Cruciani RA, Barker JL, Zasloff M, et al. Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. Proc. Natl. Acad. Sci. USA. 1991, 88:3792-3796
24. Hobta A, Lisovskiy I, Mikhalap S, et al. Epidermoid carcinoma-derived antimicrobial peptide (ECAP) inhibits phosphorylation by protein kinases in vitro. Cell Biochem Funct. 2001, 19(4) :291-298
25、 王芳,张双全,戴祝英.抗菌肽CM4组分对K562癌细胞染色 质DNA断裂作用的SCGE研究.生物化学与生物物理进展. 1998,25(1) :64-67
26、 贾世荣,屈贤铭主编.马铃薯抗菌肽基因工程.北京:中国农业 科技出版社1996,75-80
27、 贾红武,张双全,戴祝英.家蚕抗菌肽对癌细胞的杀伤作用及其
超微结构的观察.动物学研究.1996,18(3) :325-331
28、 张双全,贾红武,戴祝英.抗菌肽CM4抗K562癌细胞的超微 结构研究.生物化学与生物物理进展.1997,24(2) :159-163
29、 王芳,张双全,杨伟平.抗菌肽CM4组分对离体癌细胞的影响. 南京师大学报(自然科学版).1 998,2 1(4) :53-57
30. Shin SY, Kang JH,Jang SY, et al. Effects of the hinge region of cecropin A91-8) -magainin 2(1-12) , a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells. Biochimica et Biophysica Acta. 2000,1463:209-218
31. Haimovich B, Tanaka JC. Magainin-induced cytotoxicity in eukaryotic cells :kinetics, dose-response and channel characteristics. Biochim Biophys Acta. 1995,1240(2) : 149-158
32. Srisailam S, Kumar TK, Arunkumar AI, et al. Crumpled structure of the custom hydrophobic lytic peptide cecropin B3. Eur J Biochem. 2001,268(15) :4278-4284
33. Chen Y, Xu X,Hong S,et al. RGD-Tachyplesin inhibits tumor growth. Cancer Research. 2001,61:2434-2438
34、 许玉澄,张双全,戴祝英.家蚕抗菌肽的抗癌作用.动物学研究. 1 998. 1 9(4) :263-268
35. Yoo YC, Watanabe R, Koike Y, et al. Apoptosis in human leukemic cells induced by lactoferricin, a bovine milk protein-derived peptide:involvement of reactive oxygen species. Biochem Biophys Res Commun. 1997,237(3) 624-628
36. Ellerby HM, Arap W, Ellerby LM et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nature Medcine. 1999,5(9) : 1032-1038
37. Mai JC, Mi Z, Kim SH, et al. A proapoptotic peptide for the treatment of solid tumors. Cancer Res. 2001, 61(21) :7709-7712
38. Betten A, Bylund J, Cristophe T et al. A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocute dysfunction and apoptosis. J.Clin.Invest. 2001, 108: 1221-1228
39. Ohtake T, Fujimoto Y, Ikuta K, et al. Proline-rich antimicrobial peptide, PR-39 gene transduction altered invasive activity and actin structure in human hepatocellular carcinoma cells. Br J Cancer. 1999,81(3) :393-403
40. Tanaka K, Fujinmoto Y, Suzuki M, et al. PI3-kinase p85alpha is a target molecule of proline-rich antimicrobial peptide to suppress proliferation of ras-transformed cells. Jpn J Cancer Res. 2001,92(9) :959-967
41. Yoo YC, Watanabe S, Watanabe R, et al. Bovine lactoferrin and lactoferricin inhibit tumor metastasis in mice. Adv Exp Med Biol. 1998,443:285-291
42. ligo M, Kuhara T, Ushida Y,et al. Inhibitory effects of bovine lactoferrin on colon carcinoma 26 lung metastasis in mice. Clin Exp Metastasis. 1999,17(1) :35-40
43. Moore AJ, Devine DA, Bibby MC. Preliminary experimental anticancer activity of cecropins. Pept Res. 1994,7(5) :265-269
44. Baker MA, Maloy WL, Zasloff M, et al. Anticancer efficacy of magainin2 and analogue peptides. Cancer Res. 1993,53(13) :3052-3057
45. Soballe PW, Maloy WL, Myrga ML, et al. Experimental local therapy of human melanoma with lytic magainin peptides. Int J Cancer. 1995,60(2) :280-284
46、 郭玉梅,戴祝英,胡云龙.家蚕抗菌肽的一些性质及抗肿瘤活 性.南京师范大学学报(自然科学版).1995,18(1) :62-67
47、 王芳,张双全.抗菌肽抗癌作用机理的研究进展.自然科学 进展.1999,9(2) :97-102
48. Tsuda H, Sekine K, Nakamura J, et al. Inhibition of azoxymethane initiated colon tumor and aberrant crypt foci development by bovine lactoferrin administration in F344 rats. Adv Exp Med Biol. 1998, 443:273-284
49. Winder D, Gunzburg WH, Erfle V,et al. Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem Biophys Res Commun.1998, 242(3) :608-612
50. Prenner EJ, Lewis N.A.H., Jelokhani-Niaraki M, et al. Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes. Biochimica et Biophysica Acta. 2001,1510:83-92
51. Chan SC, Hui L,Chen HM. Enhancement of the cytolytic effect of anti-bacterial cecropin by the microvilli of cancer cells. Anticancer Res. 1998,18(6A):4467-4474
52. Nakamura T, Furunaka H, Miyata T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (tachypleus tridentatus). The Journal of Biological Chemistry. 1988,263:16709-16713
53. Maloy WL, Kari UP. Structure-activity studies on magainins and other host defense peptides. Biopolymers 1995, 37(2) : 105-122
54. Matsuzaki K, Sugishita K, Harada M, et al. Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria. Biochim. Biophys. Acta. 1997,1327:119-130
55. Kobayashi S, Hirakura Y, Matsuzaki K..Bacteria-selective synergism between the antimicrobial peptides alpha-helical
magainin 2 and cyclic beta-sheet tachyplesin Ⅰ: toward cocktail therapy Biochemistry. 2001,40(48) : 14330-14335
56. Miyata T, Tokunaga F, Yoneya T, et al. Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin Ⅱ, and polyphemusins Ⅱ andⅡ: chemical structures and biological activity. J. Biochem. 1989, 106(4) :663-668
57. Tamamura H, Ikoma R, Niwa M, et al. Antimicrobial activity and conformation of tachyplesin Ⅰ and its analogs Chem. Pharm. Bull. 1993,41(5) :978-980
58. Matsuzaki K, Fukui M, Fujii N, et al. Interaction of an antimicrobial peptide, tachyplesin-Ⅰ, with lipid-membranes. Biochimica et Biophysica Acta. 1991,1070(1) :259-264
59. Katsu T, Nakao S, Iwanaga S. Mode of action of an antimirobial peptide, tachyplesin-Ⅰ, on biomembranes:Biological and pharmaceutical Bulletin. 1993, 16:178-181
60. Masuda K, Ohta M, Ito M, et al. Bactericidal action of tachyplesin-Ⅰ against oral streptococci. Oral Microbiology And Immunology. 1994,9:77-80
61. Ga S, Yamashita S, Ohno M. A novel anti-HIV synthetic peptide, t-22([tyr5,12,lys7] -polyphemusin-Ⅱ).Biochemistry. 1992,31:122 41-12247
62. Matsuzaki K, Fukui M,Fujii N, et al. Permeabilization and morphological-changes in phosphatidylglycerol bilayers induced by an antimicrobial peptide,tachyplesin-Ⅱ. Collod and Polymer science. 1993,271:901-908
63. Matsuzaki K, Yoneyama S, Fujii N, et al. Memberane permeabilization mechanisms of a cyclic antimicrobial peptide,tachypplesin I, and its linear analog. Biochemistry.
1997,36:9799-9806
64. Hirakura Y, Kobayashi S, Matsuzaki K. Specific interactions of the antimicrobial peptide cyclic β-sheet tachylesin I with lipopolysaccharides. Biochimica et Biophysica Acta 2002,1562:32-36
65. Cotter TG, Lennon SV, Glynn JG et al. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumour and normal cells. Anticancer Res. 1990,10(5A): 1153-1159
66. Degos L. A new concept: treatment using differentiation of malignant cell in man. Bull Acad Natl Med. 1995,179(8) : 1689-1700
67. Heller WT, Waring AJ, Lehrer RI, et al.. Multiple states of β-sheet peptide protegrin in lipid bilayers. Biochemistry. 1998, 37:17331-17338
68. Pouny Y, Rapapot D, Mor A et al. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry. 1992, 31(49) :12416-12423
69. Prenne EJ, Lewis RN and Elhaney RN. The interaction of the antimicrobial peptide gramicidin S with lipid bilaye model and biological membranes. Biochim. Biophys. Acta. 1999, 1462:201-221
70. Yonezawa A, Kuwahara J, Fujii N, et al. Binding of tachyplesin-I to DNA revealed by footprinting analysis-significant aontribution of secondary structure to NA-binding and implication for biological. Biochemistry 1992, 31(11) :2998-3004
71. Rao AG. Conformation and antimicrobial activity of linear derivatives of tachyplesin lacking disulfide bonds. Arch. Biochem. Biophys. 1999, 361(1) : 127-134
72. Tarn JP, Wu CW, Yang JL. Membranolytic selectivity of cystine-stabilized cyclic protegrins. European journal of biochemisty. 2000,267(11) :3289-3300
73. Krishnakumari V. Sharadadevi A, Sitaram N, et al. Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide. Journal of Peptide Research. 1999,54(6) :528-535
74. Skerlavaj B, Gennaro R, Bagella L, et al. Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. The Journal of Biological Chemistry. 1996,271:28375-28381
75. Shin SY, Lee SH, Yang ST, et al. Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.J Pept Res. 2001,58(6) :504-514
76. Oh D, Shin SY, Lee S, et al. Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8) -magainin2(1-12) and its analogues, on their antibiotic activities and structures. Biochemistry. 2000,39(39) : 11855-11864
77. Shi SY, Kang JH, Hahm KS. Structure-antibacterial, antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides. J Pept Res. 1999,53(1) :82-90