XPNPEP2介导肿瘤导向多肽TMTP1入胞并选择性诱导肿瘤细胞凋亡的分子机理研究
摘要
目的：寻找、验证肿瘤靶向多肽TMTP1的受体，并对TMTP1靶向肿瘤作用的分子机理做初步探讨，寻找肿瘤治疗可能的新靶点。
方法：利用Pulldown技术寻找TMTP1可能的受体，通过逆向亲和实验、生物信息学分析、竞争拮抗实验以及Realtime PCR和Western等方法从受体与配体的结合、受体的分布以及功能效应等方面加以验证；以MTT、FCM、Western-blot等方法验证TMTP1具有诱导凋亡以抑制肿瘤细胞生长及增殖的生物作用，并探讨TMTP1与受体结合后，发挥TMTP1生物效应的分子机理。
结果：XPNPEP2和Hsp27是利用Pulldown技术寻找到的TMTP1可能的受体。其中XPNPEP2存在于细胞膜表面，可以和TMTP1稳固结合，并可以显著影响TMTP1与肿瘤细胞亲和和诱导凋亡的能力，其细胞、组织的表达分布也与TMTP1的筛选原理想吻合；Hsp27在细胞内与TMTP1结合，通过PI3K／Hsp27信号通路拮抗TMTP1诱导肿瘤细胞凋亡的生物学作用。
结论：XPNPEP2是TMTP1的细胞膜受体，并介导了TMTP1进入肿瘤细胞内部；Hsp27是TMTP1的胞内结合蛋白，在拮抗TMTP1诱导肿瘤细胞凋亡过程中起着关键作用。
Objective:To capture and verify the receptor of a tumor homing peptide TMTP1 is helpfulto know about the mechanism of tumor homing effect.The receptor may be a potentialtherapeutic target.
Methods:The motheds of pull-down (affinity chromatography)method were used toseparate the membrane protein molecules interacting with TMTP1 and indetify the somekind(s)of protein(s)using the Spectral analysis from the mixture of proteins.The validationof receptor including following aspect:the subcellular localization,the distribution,thebinding and the functional testing.We also used MTT assay,FCM,western-blot to verifythe inhibiton on tumor cell proliferation induced by TMTP1 and to investigate themechanism.
Results:XPNPEP2 and Hsp27 are candidate receptor captured by pull-down.XPNPEP2expresses in the cellular surface,it can bind TMTP1 strongly and shows a good results inthe functional test.Its distribution also coincides with the rules which we screens theTMTP1 by phage display.Hsp27 is a intracellular binding protein of TMTP1,it negativelyregulate the apoptosis induced by TMTP1 through the PI3K/Hsp27 pathway.
Conclusions:XPNPEP2 is a cellular surface receptor of TMTP1 and mediate theendocytosis of TMTP1.Hsp27 is a intracellular binding protein of TMTP 1,it plays a keyrole in the apoptosis induced by TMTP 1.
方法：利用Pulldown技术寻找TMTP1可能的受体，通过逆向亲和实验、生物信息学分析、竞争拮抗实验以及Realtime PCR和Western等方法从受体与配体的结合、受体的分布以及功能效应等方面加以验证；以MTT、FCM、Western-blot等方法验证TMTP1具有诱导凋亡以抑制肿瘤细胞生长及增殖的生物作用，并探讨TMTP1与受体结合后，发挥TMTP1生物效应的分子机理。
结果：XPNPEP2和Hsp27是利用Pulldown技术寻找到的TMTP1可能的受体。其中XPNPEP2存在于细胞膜表面，可以和TMTP1稳固结合，并可以显著影响TMTP1与肿瘤细胞亲和和诱导凋亡的能力，其细胞、组织的表达分布也与TMTP1的筛选原理想吻合；Hsp27在细胞内与TMTP1结合，通过PI3K／Hsp27信号通路拮抗TMTP1诱导肿瘤细胞凋亡的生物学作用。
结论：XPNPEP2是TMTP1的细胞膜受体，并介导了TMTP1进入肿瘤细胞内部；Hsp27是TMTP1的胞内结合蛋白，在拮抗TMTP1诱导肿瘤细胞凋亡过程中起着关键作用。
Objective:To capture and verify the receptor of a tumor homing peptide TMTP1 is helpfulto know about the mechanism of tumor homing effect.The receptor may be a potentialtherapeutic target.
Methods:The motheds of pull-down (affinity chromatography)method were used toseparate the membrane protein molecules interacting with TMTP1 and indetify the somekind(s)of protein(s)using the Spectral analysis from the mixture of proteins.The validationof receptor including following aspect:the subcellular localization,the distribution,thebinding and the functional testing.We also used MTT assay,FCM,western-blot to verifythe inhibiton on tumor cell proliferation induced by TMTP1 and to investigate themechanism.
Results:XPNPEP2 and Hsp27 are candidate receptor captured by pull-down.XPNPEP2expresses in the cellular surface,it can bind TMTP1 strongly and shows a good results inthe functional test.Its distribution also coincides with the rules which we screens theTMTP1 by phage display.Hsp27 is a intracellular binding protein of TMTP1,it negativelyregulate the apoptosis induced by TMTP1 through the PI3K/Hsp27 pathway.
Conclusions:XPNPEP2 is a cellular surface receptor of TMTP1 and mediate theendocytosis of TMTP1.Hsp27 is a intracellular binding protein of TMTP 1,it plays a keyrole in the apoptosis induced by TMTP 1.
引文
1,陈竺主编《全国第三次死因回顾抽样调查报告》
2,Ravd IN PM, Cron IN KA, Howlader N. et al. The decrease in breast2cancer incidence in 2003 in the United States. N Engl J Med, 2007, 356 (16): 1670-1674.
3,Llovet J, Ricci S, Mazzaferro V, et al. Sorafenib improves survival in advanced hepatocellular carcinoma ( HCC) :Results of a phase ***** randomized placebo2controlled trial ( SHARP trial). J Clin Oncol, 2007, 25: 18 s
4,Gralow J, Ozols R F, Bajorn D F, et al. Clinical Cancer Advances 2007: Major Research Advances in Cancer Treatment, Prevention, and Screening2A Report From the American Society of clinical oncology[ J ]. J Clin Oncol, 2007, 26 (2): 313-325.
5,Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res, 2001; 7:2958- 2970
6,Chung KY. Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol, 2005; 23:1803-1810
7,Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 1998 Jan 16; 279(5349):377-380.
1. Einarson, M.B. Orlinick, J.R. (2002). Identification of Protein-Protein Interactions with Glutathione S-Transferase Fusion Proteins. In Protein-Protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, pp. 37-57.
2. Einarson, M.B. (2001). Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique. In Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, pp. 18.55-18.59.
3. Vikis, H.G. and Guan, K.-L. (2004). Glutathione-S-Transferase-Fusion Based Assays for Studying Protein-Protein Interactions. In Protein-Protein Interactions, Methods and Applications, Methods in Molecular Biology, 261, Fu, H. Ed. Humana Press, Totowa, N.J.,pp. 175-186.
4. Sprenger R R, Speijer D, Back J W, et al. Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry. electrophoresis, 2004, 25(1): 156-172
5. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R. and Matthews, B. W. Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,2473-2477
6. Molinaro, G, Boileau, G. and Adam, A. Aminopeptidase P and vasoactive peptides: from fundamental aspects to clinical interests. In Aminopeptidases in Biology and Disease (Hooper, N. M. and Lendeckel, U., eds.), 2004, pp. 251-269
7. N.M.Hooper, A.J. Turner, FEBS Lett. 229 1988.340-344.
8. J.W. Ryan, A. Papapetropoulos, H. Ju, N.D. Denslow, A.Antonov, R. Virmani, F.D. Kolodgie, R.G Gerrity, J.D. Catravas, Immunopharmacol. 32 1996.149-152.
9. Phil Oh, Per Borgstrom, Halina Witkiewicz, et al. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechnol. 2007; 25(3): 327-337.
1.Liu Y, Zheng J, Fang W, et al [Isolation and characterization of human prostate cancer cell subclones with different metastatic potential]. Zhonghua Bing Li Xue Za Zhi 1999;28: 361-4.
2.Tai SK, Tan OJ, Chow VT, Jin R, et al.Differential expression of metallothionein 1 and 2 isoforms in breast cancer lines with different invasive potential: identification of a novel nonsilent metallothionein-1H mutant variant. Am J Pathol 2003; 163(5):2009-19.
3.Kroemer G,Reed JC. Mitochondrial control of cell death. Nat Med ,2000 ,6 (5):513-519.
4.Li H , Zhu H , Xu CJ , et al . Cleavage of BID by caspase28 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 1998 , 94 (4): 491-501.
5.Rao RV ,Hermel E ,Castro2Obregon S , et al . Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation. J Biol Chem ,2001 ,276 (36) : 33869-33874.
6.Yang W, Luo D, Wang S, Wang R, et al.TMTP1, a novel tumor-homing peptide specifically targeting metastasis. Clin Cancer Res. 2008; 14(17):5494-502
7.Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 1993; 27: 437-496.
8.Samali A, Orrenius S. Heat shock proteins: Regulators of stress response and apoptosis. Cell Stress and Chaperones 1998; 3: 228-236.
9.Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 1992; 313: 307-313.
10.Kulik G, Klippel A,Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Molecular and Cellular Biology 1997; 17:1595-1606.
11. Konishi H, Matsuzaki H, Tanaka M, et al. Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27. FEBS Lettl997; 410: 493-498.
1.Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 1993; 27: 437-496.
2.Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet 1988; 22: 631-677.
3.Samali A, Orrenius S. Heat shock proteins: Regulators of stress response and apoptosis. Cell Stress and Chaperones 1998; 3: 228-236.
4.Morino M, Tsuzuki T, Ishikawa Y, et al. Specific expression of HSP27 in human tumor cell lines in vitro.In Vivo , 1997 ,11 (2):179-184.
5.De Jong WW, Leunissen JA, Voorter CE. Evolution of the alpha-crystallm/small heat-shock protein family. Molecular Biology and Evolution 1993; 10: 103-126.
6.Landry J, Lambert H, Zhou M, et al. Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogenactivated kinases that recognize the same amino acid motif as S6 kinase Ⅱ. J Biol Chem 1992; 267: 794-803.
7.Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 1992; 313: 307-313.
8.Freshney NW, Rawlinson L, Guesdon F, et al. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 1994; 78: 1039-1049.
9.Landry J, Chretien P, Laszlo A, Lambert H. Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells. J Cell Physiol 1991; 147:93-101.
10.Pauli D, ArrigoAP,Vazquez J,Tonka CH,Tissieres A. Expression of the small heat shock genes during Drosophila development: Comparison of the accumulation of hsp23 and hsp27mRNAs and polypeptides. Genome 1989; 31: 671-676.
11.Soldatenkov VA, Dritschilo A. Apoptosis of Ewing's sarcoma cells is accompanied by accumulation of ubiquitinated proteins. Cancer Res 1997; 57: 3881-3885.
12. Jakob U, Gaestel M, Engel K, Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem 1993; 268:1517-1520.
13. Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986; 232: 522-524.
14. DiDomenico BJ, Bugaisky GE, Lindquist S. The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels. Cell 1982; 31: 593-603.
15. Garrido C, Bruey JM, Fromentin A, Hammann A, Arrigo AP, Solary E. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J 1999; 13: 2061-2070.
16. Samali A, Robertson JD, Peterson E, et al. Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli.Cell Stress and Chaperones 2001; 6: 49-58.
17. Bruey JM, Ducasse C, Bonniaud P, et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol 2000; 2: 645-652.
18. Concannon CG, Orrenius S, Samali A. Hsp27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c. Gene Expr 2001; 9:195-201.
19. Pandey P, Farber R, Nakazawa A, et al. Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3. Oncogene 2000; 19: 1975-1981.
20. Polla BS, Kantengwa S, Francois D, et al. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc Natl Acad Sci USA 1996; 93: 6458-6463.
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2,Ravd IN PM, Cron IN KA, Howlader N. et al. The decrease in breast2cancer incidence in 2003 in the United States. N Engl J Med, 2007, 356 (16): 1670-1674.
3,Llovet J, Ricci S, Mazzaferro V, et al. Sorafenib improves survival in advanced hepatocellular carcinoma ( HCC) :Results of a phase ***** randomized placebo2controlled trial ( SHARP trial). J Clin Oncol, 2007, 25: 18 s
4,Gralow J, Ozols R F, Bajorn D F, et al. Clinical Cancer Advances 2007: Major Research Advances in Cancer Treatment, Prevention, and Screening2A Report From the American Society of clinical oncology[ J ]. J Clin Oncol, 2007, 26 (2): 313-325.
5,Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res, 2001; 7:2958- 2970
6,Chung KY. Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol, 2005; 23:1803-1810
7,Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 1998 Jan 16; 279(5349):377-380.
1. Einarson, M.B. Orlinick, J.R. (2002). Identification of Protein-Protein Interactions with Glutathione S-Transferase Fusion Proteins. In Protein-Protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, pp. 37-57.
2. Einarson, M.B. (2001). Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique. In Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, pp. 18.55-18.59.
3. Vikis, H.G. and Guan, K.-L. (2004). Glutathione-S-Transferase-Fusion Based Assays for Studying Protein-Protein Interactions. In Protein-Protein Interactions, Methods and Applications, Methods in Molecular Biology, 261, Fu, H. Ed. Humana Press, Totowa, N.J.,pp. 175-186.
4. Sprenger R R, Speijer D, Back J W, et al. Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry. electrophoresis, 2004, 25(1): 156-172
5. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R. and Matthews, B. W. Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,2473-2477
6. Molinaro, G, Boileau, G. and Adam, A. Aminopeptidase P and vasoactive peptides: from fundamental aspects to clinical interests. In Aminopeptidases in Biology and Disease (Hooper, N. M. and Lendeckel, U., eds.), 2004, pp. 251-269
7. N.M.Hooper, A.J. Turner, FEBS Lett. 229 1988.340-344.
8. J.W. Ryan, A. Papapetropoulos, H. Ju, N.D. Denslow, A.Antonov, R. Virmani, F.D. Kolodgie, R.G Gerrity, J.D. Catravas, Immunopharmacol. 32 1996.149-152.
9. Phil Oh, Per Borgstrom, Halina Witkiewicz, et al. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechnol. 2007; 25(3): 327-337.
1.Liu Y, Zheng J, Fang W, et al [Isolation and characterization of human prostate cancer cell subclones with different metastatic potential]. Zhonghua Bing Li Xue Za Zhi 1999;28: 361-4.
2.Tai SK, Tan OJ, Chow VT, Jin R, et al.Differential expression of metallothionein 1 and 2 isoforms in breast cancer lines with different invasive potential: identification of a novel nonsilent metallothionein-1H mutant variant. Am J Pathol 2003; 163(5):2009-19.
3.Kroemer G,Reed JC. Mitochondrial control of cell death. Nat Med ,2000 ,6 (5):513-519.
4.Li H , Zhu H , Xu CJ , et al . Cleavage of BID by caspase28 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 1998 , 94 (4): 491-501.
5.Rao RV ,Hermel E ,Castro2Obregon S , et al . Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation. J Biol Chem ,2001 ,276 (36) : 33869-33874.
6.Yang W, Luo D, Wang S, Wang R, et al.TMTP1, a novel tumor-homing peptide specifically targeting metastasis. Clin Cancer Res. 2008; 14(17):5494-502
7.Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 1993; 27: 437-496.
8.Samali A, Orrenius S. Heat shock proteins: Regulators of stress response and apoptosis. Cell Stress and Chaperones 1998; 3: 228-236.
9.Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 1992; 313: 307-313.
10.Kulik G, Klippel A,Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Molecular and Cellular Biology 1997; 17:1595-1606.
11. Konishi H, Matsuzaki H, Tanaka M, et al. Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27. FEBS Lettl997; 410: 493-498.
1.Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 1993; 27: 437-496.
2.Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet 1988; 22: 631-677.
3.Samali A, Orrenius S. Heat shock proteins: Regulators of stress response and apoptosis. Cell Stress and Chaperones 1998; 3: 228-236.
4.Morino M, Tsuzuki T, Ishikawa Y, et al. Specific expression of HSP27 in human tumor cell lines in vitro.In Vivo , 1997 ,11 (2):179-184.
5.De Jong WW, Leunissen JA, Voorter CE. Evolution of the alpha-crystallm/small heat-shock protein family. Molecular Biology and Evolution 1993; 10: 103-126.
6.Landry J, Lambert H, Zhou M, et al. Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogenactivated kinases that recognize the same amino acid motif as S6 kinase Ⅱ. J Biol Chem 1992; 267: 794-803.
7.Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 1992; 313: 307-313.
8.Freshney NW, Rawlinson L, Guesdon F, et al. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 1994; 78: 1039-1049.
9.Landry J, Chretien P, Laszlo A, Lambert H. Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells. J Cell Physiol 1991; 147:93-101.
10.Pauli D, ArrigoAP,Vazquez J,Tonka CH,Tissieres A. Expression of the small heat shock genes during Drosophila development: Comparison of the accumulation of hsp23 and hsp27mRNAs and polypeptides. Genome 1989; 31: 671-676.
11.Soldatenkov VA, Dritschilo A. Apoptosis of Ewing's sarcoma cells is accompanied by accumulation of ubiquitinated proteins. Cancer Res 1997; 57: 3881-3885.
12. Jakob U, Gaestel M, Engel K, Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem 1993; 268:1517-1520.
13. Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986; 232: 522-524.
14. DiDomenico BJ, Bugaisky GE, Lindquist S. The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels. Cell 1982; 31: 593-603.
15. Garrido C, Bruey JM, Fromentin A, Hammann A, Arrigo AP, Solary E. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J 1999; 13: 2061-2070.
16. Samali A, Robertson JD, Peterson E, et al. Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli.Cell Stress and Chaperones 2001; 6: 49-58.
17. Bruey JM, Ducasse C, Bonniaud P, et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol 2000; 2: 645-652.
18. Concannon CG, Orrenius S, Samali A. Hsp27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c. Gene Expr 2001; 9:195-201.
19. Pandey P, Farber R, Nakazawa A, et al. Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3. Oncogene 2000; 19: 1975-1981.
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