癌症的轉移是腫瘤發展過程中最難治療的致死原因。目前對於腫瘤細胞轉移與生長的新興療法是以腫瘤內血管新生的特性作為標的物，試圖透過抑制血管新生來達到抑制腫瘤生長及轉移的目的。然而，在這些進程中，腫瘤細胞與腫瘤微環境的發炎現象也扮演著一定的角色。我們利用慢病毒 (Lentivirus) 載體攜帶kallistatin基因 (LV-Kallistatin) 以進行小鼠肺癌 (LL/2) 轉移與生長的治療。在利用以LV-Kallistatin感染細胞後所取得的條件培養液與人類臍靜脈內皮細胞 (HUVEC) 或LL/2作用後發現，細胞的轉移、增生、黏附和侵噬的能力有被抑制的現象。此外，先以LV-Kallistatin感染穩定表現冷光的肺癌細胞株 (LL-Luc) 後篩選出可以穩定表現這兩種蛋白質的肺癌細胞株 (LL-Luc-Kallistatin) ，再以尾靜脈給予的方式來分析kallistatin在小鼠體內對腫瘤細胞的影響。實驗證實表現kallistatin的肺癌細胞株生長有趨緩的現象。在直接以慢病毒傳送kallistatin基因對模擬腫瘤轉移實驗模式 (experimental metastasis model) 進行治療後，從肺臟腫瘤組織的重量評估與存活期的觀察顯示了LV-Kallistatin在癌症基因治療中的扮演著重要的角色。利用酵素連結免疫檢測 (enzyme-linked immunoassay, ELISA) 及組織免疫染色 (immunohistochemistry, IHC) 的方式，我們也進一步發現與腫瘤生長、黏附至血管細胞及發炎相關的腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)、轉型生長因子-β (transforming growth factor-β, TGF-β) 的表現在給予LV-Kallistatin治療的組別中和對照組有所不同之現象，因此推測kallistatin可能會透過這類細胞激素來調控腫瘤微環境。並發現巨噬細胞 (macrophage) 在LV-Kallistatin治療組中有較少的浸潤，暗示了給予治療後腫瘤組織的發炎反應有趨緩的現象。綜合以上的實驗結果，我們認為kallistatin可能透過抑制血管新生 (anti-angiogenesis) 及調控發炎反應 (anti-inflammation) 進而對癌症產生有效的治療效果。

Induction of angiogenesis plays an important role in the development and progression of most human tumors, including lung cancer. Currently available therapies for malignant lung cancer produce low response rates in patients. Therefore, more effective treatment modalities are needed. Therapeutic targeting of angiogenesis has recently been explored to inhibit malignant tumor growth and metastasis. In this study, the expression of human gene kallistatin in Lewis Lung carcinoma cells (LL/2), resulting in inhibiting the metastatic tumor growth and prolonging the survival of tumor bearing mice. Meanwhile, we constructed lentivirus vectors carrying human gene kallistatin (LV-Kallistatin) for gene therapy. By using the conditioned medium from LV-Kallistatin infected cells, we demonstrated that migration and proliferation of endothelial cell were inhibited. The similar results were also observed in migration, invasion and adhesion of LL/2. In experimental metastatic study, the wet lung weights from animals that received LV-Kallistatin were decreased compared with control-treated mice. Besides, the survival rate was dramatically extended in LV-Kallistatin treated group. By using the Enzyme-linked immunoassay (ELISA), we analyzed the cytokine expression at the tumor site. Tumor necrosis factor-α (TNF-α) was lower in LV-Kallistatin treated group compared with control group. On the contrary, transforming growth factor-β (TGF-β) was slightly higher in LV Kallistatin treated group than the control group. According to our findings, lentivirus mediated kallistatin expression has provided a promising therapeutic potential in cancer metastasis therapy.

Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., and Muschel, R. J. (2000). Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat Med 6, 100-102.

Balkwill, F., Charles, K. A., and Mantovani, A. (2005). Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7, 211-217.

Baum, C., and von Kalle, C. (2003). Gene therapy targeting hematopoietic cells: better not leave it to chance. Acta Haematol 110, 107-109.

Bevilacqua, M. P. (1993). Endothelial-leukocyte adhesion molecules. Annu Rev Immunol 11, 767-804.

Bogdan, C., and Nathan, C. (1993). Modulation of macrophage function by transforming growth factor beta, interleukin-4, and interleukin-10. Ann N Y Acad Sci 685, 713-739.

Bogenrieder, T., and Herlyn, M. (2003). Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 22, 6524-6536.

Brigati, C., Noonan, D. M., Albini, A., and Benelli, R. (2002). Tumors and inflammatory infiltrates: friends or foes? Clin Exp Metastasis 19, 247-258.

Bussolino, F., Mantovani, A., and Persico, G. (1997). Molecular mechanisms of blood vessel formation. Trends Biochem Sci 22, 251-256.

Chao, J., Yin, H., Yao, Y. Y., Shen, B., Smith, R. S., Jr., and Chao, L. (2006). Novel role of kallistatin in protection against myocardial ischemia-reperfusion injury by preventing apoptosis and inflammation. Hum Gene Ther 17, 1201-1213.

Coussens, L. M., and Werb, Z. (2002). Inflammation and cancer. Nature 420, 860-867.
Dawson, D. W., Volpert, O. V., Gillis, P., Crawford, S. E., Xu, H., Benedict, W., and Bouck, N. P. (1999). Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285, 245-248.

de Caestecker, M. P., Piek, E., and Roberts, A. B. (2000). Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst 92, 1388-1402.

Decaussin, M., Sartelet, H., Robert, C., Moro, D., Claraz, C., Brambilla, C., and Brambilla, E. (1999). Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol 188, 369-377.

Drixler, T. A., Borel Rinkes, I. H., Ritchie, E. D., van Vroonhoven, T. J., Gebbink, M. F., and Voest, E. E. (2000). Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. Cancer Res 60, 1761-1765.

Dvorak, H. F., Brown, L. F., Detmar, M., and Dvorak, A. M. (1995). Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 146, 1029-1039.

Elgert, K. D., Alleva, D. G., and Mullins, D. W. (1998). Tumor-induced immune dysfunction: the macrophage connection. J Leukoc Biol 64, 275-290.

Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1, 27-31.

Folkman, J. (2001). Angiogenesis-dependent diseases. Semin Oncol 28, 536-542.

Folkman, J., and Haudenschild, C. (1980). Angiogenesis in vitro. Nature 288, 551-556.

Folkman, J., and Shing, Y. (1992). Angiogenesis. J Biol Chem 267, 10931-10934.
Gao, G., Shao, C., Zhang, S. X., Dudley, A., Fant, J., and Ma, J. X. (2003). Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage. Diabetologia 46, 689-698.

Hadden, J. W. (2003). Immunodeficiency and cancer: prospects for correction. Int Immunopharmacol 3, 1061-1071.

Hanahan, D., and Folkman, J. (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364.

Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.

Janes, S. M., and Watt, F. M. (2006). New roles for integrins in squamous-cell carcinoma. Nat Rev Cancer 6, 175-183.

Jonjic, N., Peri, G., Bernasconi, S., Sciacca, F. L., Colotta, F., Pelicci, P., Lanfrancone, L., and Mantovani, A. (1992). Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med 176, 1165-1174.

Karin, M. (2006). Nuclear factor-kappaB in cancer development and progression. Nature 441, 431-436.

Karin, M., and Greten, F. R. (2005). NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5, 749-759.

Klagsbrun, M., and Moses, M. A. (1999). Molecular angiogenesis. Chem Biol 6, R217-224.

Kuhn, H., Kopff, C., Konrad, J., Riedel, A., Gessner, C., and Wirtz, H. (2004). Influence of basic fibroblast growth factor on the proliferation of non-small cell lung cancer cell lines. Lung Cancer 44, 167-174.

Kulbe, H., Thompson, R., Wilson, J. L., Robinson, S., Hagemann, T., Fatah, R., Gould, D., Ayhan, A., and Balkwill, F. (2007). The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells. Cancer Res 67, 585-592.

Kuper, H., Adami, H. O., and Trichopoulos, D. (2000). Infections as a major preventable cause of human cancer. J Intern Med 248, 171-183.

Lee, J., Didier, D. N., Lockett, M. R., Scalf, M., Greene, A. S., Olivier, M., and Smith, L. M. (2007). Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays. Anal Biochem.

Liang, Z. H., Wu, P. H., Li, L., Xue, G., Zeng, Y. X., and Huang, W. L. (2004). Inhibition of tumor growth in xenografted nude mice with adenovirus-mediated endostatin gene comparison with recombinant endostatin protein. Chin Med J (Engl) 117, 1809-1814.

Lin, W. W., and Karin, M. (2007). A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 117, 1175-1183.

Luo, J. L., Maeda, S., Hsu, L. C., Yagita, H., and Karin, M. (2004). Inhibition of NF-kappaB in cancer cells converts inflammation- induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression. Cancer Cell 6, 297-305.

Luo, Y., Zhou, H., Krueger, J., Kaplan, C., Lee, S. H., Dolman, C., Markowitz, D., Wu, W., Liu, C., Reisfeld, R. A., and Xiang, R. (2006). Targeting tumor-associated macrophages as a novel strategy against breast cancer. J Clin Invest 116, 2132-2141.

Maeda, H., Okamoto, T., and Akaike, T. (1998). Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals. Biol Chem 379, 193-200.

Miao, R. Q., Murakami, H., Song, Q., Chao, L., and Chao, J. (2000). Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery. Circ Res 86, 418-424.

O'Byrne, K. J., and Dalgleish, A. G. (2001). Chronic immune activation and inflammation as the cause of malignancy. Br J Cancer 85, 473-483.

O'Reilly, M. S., Pirie-Shepherd, S., Lane, W. S., and Folkman, J. (1999). Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 285, 1926-1928.

Ohno, S., Suzuki, N., Ohno, Y., Inagawa, H., Soma, G., and Inoue, M. (2003). Tumor-associated macrophages: foe or accomplice of tumors? Anticancer Res 23, 4395-4409.

Pfeifer, A., Eigenbrod, S., Al-Khadra, S., Hofmann, A., Mitteregger, G., Moser, M., Bertsch, U., and Kretzschmar, H. (2006). Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest 116, 3204-3210.

Schoppmann, S. F., Birner, P., Stockl, J., Kalt, R., Ullrich, R., Caucig, C., Kriehuber, E., Nagy, K., Alitalo, K., and Kerjaschki, D. (2002). Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol 161, 947-956.

Seth, P. (2005). Vector-mediated cancer gene therapy: an overview. Cancer Biol Ther 4, 512-517.

Tai, K. F., Chen, P. J., Chen, D. S., and Hwang, L. H. (2003). Concurrent delivery of GM-CSF and endostatin genes by a single adenoviral vector provides a synergistic effect on the treatment of orthotopic liver tumors. J Gene Med 5, 386-398.

Thomas, C. E., Ehrhardt, A., and Kay, M. A. (2003). Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4, 346-358.

Torisu, H., Ono, M., Kiryu, H., Furue, M., Ohmoto, Y., Nakayama, J., Nishioka, Y., Sone, S., and Kuwano, M. (2000). Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and IL-1alpha. Int J Cancer 85, 182-188.

Tsung, K., Dolan, J. P., Tsung, Y. L., and Norton, J. A. (2002). Macrophages as effector cells in interleukin 12-induced T cell-dependent tumor rejection. Cancer Res 62, 5069-5075.

Wahl, L. M., and Kleinman, H. K. (1998). Tumor-associated macrophages as targets for cancer therapy. J Natl Cancer Inst 90, 1583-1584.

Wang, C. R., Chen, S. Y., Wu, C. L., Liu, M. F., Jin, Y. T., Chao, L., and Chao, J. (2005). Prophylactic adenovirus-mediated human kallistatin gene therapy suppresses rat arthritis by inhibiting angiogenesis and inflammation. Arthritis Rheum 52, 1319-1324.

Wang, M. Y., Day, J., Chao, L., and Chao, J. (1989). Human kallistatin, a new tissue kallikrein-binding protein: purification and characterization. Adv Exp Med Biol 247B, 1-8.

Witham, T. F., Villa, L., Yang, T., Pollack, I. F., Okada, H., Robbins, P. D., and Chambers, W. H. (2003). Expression of a soluble transforming growth factor-beta (TGFbeta) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo. J Neurooncol 64, 63-69.

Woodhouse, E. C., Chuaqui, R. F., and Liotta, L. A. (1997). General mechanisms of metastasis. Cancer 80, 1529-1537.

Wu, Y., Zhong, Z., Huber, J., Bassi, R., Finnerty, B., Corcoran, E., Li, H., Navarro, E., Balderes, P., Jimenez, X., et al. (2006). Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer. Clin Cancer Res 12, 6573-6584.

Zhang, M., Volpert, O., Shi, Y. H., and Bouck, N. (2000). Maspin is an angiogenesis inhibitor. Nat Med 6, 196-199.

Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15, 871-875.