神經纖毛蛋白質-1為一多功能的協同受體，其可促進腫瘤的發生例如血管新生、腫瘤的生長和細胞的侵襲和轉移。而在近期的研究也被指出神經纖毛蛋白質-1在非小細胞肺癌病人中若表現量較高，病人會有較短的存活率和較快的腫瘤復發時間。另外，適體為脫氧核糖核酸或核糖核酸的單股短片段小分子，利用其摺疊形成特定的立體構型能夠專一性的結合在目標上。而他們是透過一種體外篩選的方法，指數富集的配體系統進化(SELEX)篩選出來所獲得的。而其高度專一性的適體具有良好的診斷與治療應用的潛力，因此，藉由篩選出來的特定神經纖毛蛋白質-1適體將可能成為非小細胞癌症患者的一種治療方法。首先，將特定神經纖毛蛋白-1適體透過膜指數富集的配體系統進化篩選出來，在進一步使用蛋白質分析儀計算出適體之解離常數，確定其適體與神經纖毛蛋白-1有高度結合專一性。接著為了確認特定神經纖毛蛋白-1適體能否影響細胞內之功能，包括了腫瘤細胞的細胞爬行與內皮細胞之血管生成之能力。透過計算其解離常數而取得AP5、AP15和AP37這三條對神經纖毛蛋白-1有高度親和力之適體。對於神經纖毛蛋白-1在肺腺癌細胞株CL1-5中的功能的影響，特定神經纖毛蛋白-1適體降低了非小細胞肺癌細胞之細胞爬行和侵犯能力，此外對於癌細胞並無任何毒殺效果。另外，臍靜脈內皮細胞在加入特定神經纖毛蛋白-1適體之後，亦降低了細胞爬行和血管生成之能力，且對於臍靜脈內皮細胞也無毒殺能力。以上的結果說明了神經纖毛蛋白-1適體能夠降低腫瘤細胞的爬行、侵犯和血管新生的能力。另外，我們將研究種點放於神經纖毛蛋白-1適體中的AP15，證明其AP15與肺腺癌細胞株CL1-5和神經纖毛蛋白-1過度表現之肺腺癌細胞株CL1-0細胞中的神經纖毛蛋白-1有直接的結合的現象。更發現AP15之功能區域之序列– AP15M3，AP15M3亦能有效降低臍靜脈內皮細胞之血管新生能力和肺腺癌細胞株CL1-5之細胞爬行能力。以上結果說明神經纖毛蛋白-1適體可以當作極具潛力的肺癌標靶治療藥物。

Neuropilin 1 (NRP1) is a multifunctional co-receptor and promotes cancer progression, including angiogenesis, tumor growth, tumor invasion and metastasis. It is reported that non-small cell lung cancer (NSCLC) patients with high expression of NRP1 have shorter disease-free and overall survival in recent researches. On the other hand, aptamers are single-stranded DNA or RNA molecules with special conformational features, efficiently binding to their targets. They are obtained by a particular in vitro selection strategy, systematic evolution of ligands by exponential enrichment (SELEX). Recently, aptamers have emerged as a novel and powerful class of ligands with excellent potential for diagnostic and therapeutic applications. Thus, development of specific aptamers against NRP1 would be a promising therapy for NSCLC patients. First, we screened and isolated NRP1-specific aptamers by membrane SELEX. Second, we determined the binding affinities of NRP1-specific aptamers binding from NRP1 by a surface plasmon resonance analysis. Then, we examined the effect of NRP1-specific aptamers on the cellular function of NRP1, containing cell migration of tumor cells and tube formation of endothelial cells. We obtained three NRP1-specific aptamers, AP5, AP15 and AP37 that had high affinities for NRP1. On the functions of NRP1 in cancer cells, NRP1-specific aptamers decreased the migration and invasion abilities of highly invasive NSCLC cells, CL1-5 cells but did not have any cytotoxicity in the cells compared with scramble-aptamers. Moreover, NRP1-specific aptamers also reduced migration and tube formation capabilities of HUVECs as well as had no cytotoxicity in HUVECs. Moreover, we proved that AP15 directly bound to NRP1 in CL1-0 cells overexpressing NRP1, and discovered the functional region of AP15, AP15M3. AP15M3, a 5’- and 3’- truncated AP15 mutant, reduced tube formation ability in HUVECs and migration ability of CL1-5 cells. These results showed that NRP1-specific aptamers decrease the migration and invasion abilities of tumor cells and angiogenesis of endothelial cells. It suggests that NRP1-specific aptamer may be a potential target therapy in lung cancer.

Arjonen, A., Kaukonen, R., and Ivaska, J. (2011). Filopodia and adhesion in cancer cell motility. Cell adhesion & migration 5, 421-430.
Bagalkot, V., Zhang, L., Levy-Nissenbaum, E., Jon, S., Kantoff, P.W., Langer, R., and Farokhzad, O.C. (2007). Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer. Nano letters 7, 3065-3070.
Ball, S.G., Bayley, C., Shuttleworth, C.A., and Kielty, C.M. (2010). Neuropilin-1 regulates platelet-derived growth factor receptor signalling in mesenchymal stem cells. The Biochemical journal 427, 29-40.
Bates, P.J., Laber, D.A., Miller, D.M., Thomas, S.D., and Trent, J.O. (2009). Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Experimental and molecular pathology 86, 151-164.
Broholm, H., and Laursen, H. (2004). Vascular endothelial growth factor (VEGF) receptor neuropilin-1's distribution in astrocytic tumors. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 112, 257-263.
Cao, S., Yaqoob, U., Das, A., Shergill, U., Jagavelu, K., Huebert, R.C., Routray, C., Abdelmoneim, S., Vasdev, M., Leof, E., et al. (2010). Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells. The Journal of clinical investigation 120, 2379-2394.
Chiu, H.F., Cheng, M.H., Tsai, S.S., Wu, T.N., Kuo, H.W., and Yang, C.Y. (2006). Outdoor air pollution and female lung cancer in Taiwan. Inhalation toxicology 18, 1025-1031.
Couraud, S., Zalcman, G., Milleron, B., Morin, F., and Souquet, P.J. (2012). Lung cancer in never smokers--a review. European journal of cancer (Oxford, England : 1990) 48, 1299-1311.
Farokhzad, O.C., Cheng, J., Teply, B.A., Sherifi, I., Jon, S., Kantoff, P.W., Richie, J.P., and Langer, R. (2006a). Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proceedings of the National Academy of Sciences of the United States of America 103, 6315-6320.
Farokhzad, O.C., Karp, J.M., and Langer, R. (2006b). Nanoparticle-aptamer bioconjugates for cancer targeting. Expert opinion on drug delivery 3, 311-324.
Ferrari, M. (2005). Cancer nanotechnology: opportunities and challenges. Nature reviews. Cancer 5, 161-171.
Gagnon, M.L., Bielenberg, D.R., Gechtman, Z., Miao, H.Q., Takashima, S., Soker, S., and Klagsbrun, M. (2000). Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity. Proceedings of the National Academy of Sciences of the United States of America 97, 2573-2578.
Glinka, Y., Mohammed, N., Subramaniam, V., Jothy, S., and Prud'homme, G.J. (2012). Neuropilin-1 is expressed by breast cancer stem-like cells and is linked to NF-kappaB activation and tumor sphere formation. Biochemical and biophysical research communications 425, 775-780.
Gold, L. (1995). Oligonucleotides as research, diagnostic, and therapeutic agents. The Journal of biological chemistry 270, 13581-13584.
Grandclement, C., and Borg, C. (2011). Neuropilins: a new target for cancer therapy. Cancers 3, 1899-1928.
Gu, C., Rodriguez, E.R., Reimert, D.V., Shu, T., Fritzsch, B., Richards, L.J., Kolodkin, A.L., and Ginty, D.D. (2003). Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Developmental cell 5, 45-57.
Ha, H., Lee, J.H., Kim, H.N., Kwak, H.B., Kim, H.M., Lee, S.E., Rhee, J.H., Kim, H.H., and Lee, Z.H. (2008). Stimulation by TLR5 modulates osteoclast differentiation through STAT1/IFN-beta. Journal of immunology (Baltimore, Md. : 1950) 180, 1382-1389.
Herbst, R.S., Heymach, J.V., and Lippman, S.M. (2008). Lung cancer. The New England journal of medicine 359, 1367-1380.
Hong, T.M., Chen, Y.L., Wu, Y.Y., Yuan, A., Chao, Y.C., Chung, Y.C., Wu, M.H., Yang, S.C., Pan, S.H., Shih, J.Y., et al. (2007). Targeting neuropilin 1 as an antitumor strategy in lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 4759-4768.
Hsieh, S.H., Ying, N.W., Wu, M.H., Chiang, W.F., Hsu, C.L., Wong, T.Y., Jin, Y.T., Hong, T.M., and Chen, Y.L. (2008). Galectin-1, a novel ligand of neuropilin-1, activates VEGFR-2 signaling and modulates the migration of vascular endothelial cells. Oncogene 27, 3746-3753.
Jayasena, S.D. (1999). Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clinical chemistry 45, 1628-1650.
Jhaveri, S., and Ellington, A. (2002). In vitro selection of RNA aptamers to a small molecule target. Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] Chapter 9, Unit 9.5.
Jia, H., Cheng, L., Tickner, M., Bagherzadeh, A., Selwood, D., and Zachary, I. (2010). Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity. British journal of cancer 102, 541-552.
Jubb, A.M., Strickland, L.A., Liu, S.D., Mak, J., Schmidt, M., and Koeppen, H. (2012). Neuropilin-1 expression in cancer and development. The Journal of pathology 226, 50-60.
Kim, D., Jeong, Y.Y., and Jon, S. (2010). A drug-loaded aptamer-gold nanoparticle bioconjugate for combined CT imaging and therapy of prostate cancer. ACS nano 4, 3689-3696.
Klinman, D.M. (2004). Immunotherapeutic uses of CpG oligodeoxynucleotides. Nature reviews. Immunology 4, 249-258.
Koch, A.E., and Distler, O. (2007). Vasculopathy and disordered angiogenesis in selected rheumatic diseases: rheumatoid arthritis and systemic sclerosis. Arthritis research & therapy 9 Suppl 2, S3.
Lai, W.Y., Wang, W.Y., Chang, Y.C., Chang, C.J., Yang, P.C., and Peck, K. (2014). Synergistic inhibition of lung cancer cell invasion, tumor growth and angiogenesis using aptamer-siRNA chimeras. Biomaterials 35, 2905-2914.
Lantuejoul, S., Constantin, B., Drabkin, H., Brambilla, C., Roche, J., and Brambilla, E. (2003). Expression of VEGF, semaphorin SEMA3F, and their common receptors neuropilins NP1 and NP2 in preinvasive bronchial lesions, lung tumours, and cell lines. The Journal of pathology 200, 336-347.
Latil, A., Bieche, I., Pesche, S., Valeri, A., Fournier, G., Cussenot, O., and Lidereau, R. (2000). VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms. International journal of cancer. Journal international du cancer 89, 167-171.
Lee, J.H., Lee, K., Moon, S.H., Lee, Y., Park, T.G., and Cheon, J. (2009). All-in-one target-cell-specific magnetic nanoparticles for simultaneous molecular imaging and siRNA delivery. Angewandte Chemie (International ed. in English) 48, 4174-4179.
Lee, Y.J., Kim, I.S., Park, S.A., Kim, Y., Lee, J.E., Noh, D.Y., Kim, K.T., Ryu, S.H., and Suh, P.G. (2013). Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis. Molecular therapy : the journal of the American Society of Gene Therapy 21, 1004-1013.
Levitzki, A. (1990). Tyrphostins--potential antiproliferative agents and novel molecular tools. Biochemical pharmacology 40, 913-918.
Li, X., Zhao, Q., and Qiu, L. (2013). Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy. Journal of controlled release : official journal of the Controlled Release Society 171, 152-162.
Li Xu, Z.Z., Zilong Zhao, Qiaoling Liu, Weihong Tan, and Xiaohong Fang (2012). Cellular Internalization and Cytotoxicity of Aptamers Selected from Lung Cancer Cell. American Journal of Biomedical Sciences 5, 47-58.
Mac Gabhann, F., and Popel, A.S. (2006). Targeting neuropilin-1 to inhibit VEGF signaling in cancer: Comparison of therapeutic approaches. PLoS computational biology 2, e180.
Machesky, L.M. (2008). Lamellipodia and filopodia in metastasis and invasion. FEBS letters 582, 2102-2111.
Marolt U, C.A., Gorenjak M, and Potrc S (2012). Generating Aptamers for Cancer Diagnosis and Therapy. Clinical and Experimental Pharmacology 2, 1-8.
Mascini, M. (2009). Aptmaers in Bioanalysis.
Matsushita, A., Gotze, T., and Korc, M. (2007). Hepatocyte growth factor-mediated cell invasion in pancreatic cancer cells is dependent on neuropilin-1. Cancer research 67, 10309-10316.
Mayer, G. (2009). The chemical biology of aptamers. Angewandte Chemie (International ed. in English) 48, 2672-2689.
McKeague, M., and Derosa, M.C. (2012). Challenges and opportunities for small molecule aptamer development. Journal of nucleic acids 2012, 748913.
Miao, H.Q., Lee, P., Lin, H., Soker, S., and Klagsbrun, M. (2000). Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14, 2532-2539.
Murga, M., Fernandez-Capetillo, O., and Tosato, G. (2005). Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 105, 1992-1999.
Nakamura, F., and Goshima, Y. (2002). Structural and functional relation of neuropilins. Advances in experimental medicine and biology 515, 55-69.
Ng, E.W., Shima, D.T., Calias, P., Cunningham, E.T., Jr., Guyer, D.R., and Adamis, A.P. (2006). Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature reviews. Drug discovery 5, 123-132.
Nimjee, S.M., Rusconi, C.P., and Sullenger, B.A. (2005). Aptamers: an emerging class of therapeutics. Annual review of medicine 56, 555-583.
Okugawa, S., Ota, Y., Kitazawa, T., Nakayama, K., Yanagimoto, S., Tsukada, K., Kawada, M., and Kimura, S. (2003). Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages. American journal of physiology. Cell physiology 285, C399-408.
Parikh, A.A., Fan, F., Liu, W.B., Ahmad, S.A., Stoeltzing, O., Reinmuth, N., Bielenberg, D., Bucana, C.D., Klagsbrun, M., and Ellis, L.M. (2004). Neuropilin-1 in human colon cancer: expression, regulation, and role in induction of angiogenesis. The American journal of pathology 164, 2139-2151.
Peng, Y., Liu, Y.M., Li, L.C., Wang, L.L., and Wu, X.L. (2014). MicroRNA-338 inhibits growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. PloS one 9, e94422.
Piechnik, A., Dmoszynska, A., Omiotek, M., Mlak, R., Kowal, M., Stilgenbauer, S., Bullinger, L., and Giannopoulos, K. (2013). The VEGF receptor, neuropilin-1, represents a promising novel target for chronic lymphocytic leukemia patients. International journal of cancer. Journal international du cancer 133, 1489-1496.
Seo, I.A., Lee, H.K., Shin, Y.K., Lee, S.H., Seo, S.Y., Park, J.W., and Park, H.T. (2009). Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130. The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology 13, 131-138.
Siegel, R., Ma, J., Zou, Z., and Jemal, A. (2014). Cancer statistics, 2014. CA: A Cancer Journal for Clinicians 64, 9-29.
Stephenson, J.M., Banerjee, S., Saxena, N.K., Cherian, R., and Banerjee, S.K. (2002). Neuropilin-1 is differentially expressed in myoepithelial cells and vascular smooth muscle cells in preneoplastic and neoplastic human breast: a possible marker for the progression of breast cancer. International journal of cancer. Journal international du cancer 101, 409-414.
Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G., and Fujisawa, H. (1991). The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors. Neuron 7, 295-307.
Treuel, L., Jiang, X., and Nienhaus, G.U. (2013). New views on cellular uptake and trafficking of manufactured nanoparticles. Journal of the Royal Society, Interface / the Royal Society 10, 20120939.
Tuerk, C., and Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science (New York, N.Y.) 249, 505-510.
Zhang, J., Liu, B., Liu, H., Zhang, X., and Tan, W. (2013). Aptamer-conjugated gold nanoparticles for bioanalysis. Nanomedicine (London, England) 8, 983-993.
Zhang, S., Zhau, H.E., Osunkoya, A.O., Iqbal, S., Yang, X., Fan, S., Chen, Z., Wang, R., Marshall, F.F., Chung, L.W., et al. (2010). Vascular endothelial growth factor regulates myeloid cell leukemia-1 expression through neuropilin-1-dependent activation of c-MET signaling in human prostate cancer cells. Molecular cancer 9, 9.
Zhang, X., Yin, P., Di, D., Luo, G., Zheng, L., Wei, J., Zhang, J., Shi, Y., Zhang, J., and Xu, N. (2009). IL-6 regulates MMP-10 expression via JAK2/STAT3 signaling pathway in a human lung adenocarcinoma cell line. Anticancer research 29, 4497-4501.