摘要
结直肠癌是最常见的恶性肿瘤之一,占消化道恶性肿瘤的第二位。结直肠癌治疗失败的主要原因是癌细胞发生了血行或淋巴转移。研究显示,结直肠癌的转移不仅与癌细胞的侵袭能力有关,还与肿瘤内血管、淋巴管的新生密切相关。大量资料表明,在人类结直肠癌中存在着血管、淋巴管的新生,而且血管、淋巴管的密度与肿瘤淋巴结转移及预后相关。抑制血管新生可以减少肿瘤的血供,诱导肿瘤细胞的凋亡,减少转移的机率;抑制淋巴管新生可以降低淋巴结转移率,改善预后。寻找有效的治疗靶点正在成为该领域的一个研究热点,其中肝细胞生长因子HGF尤其引人注目。
肝细胞生长因子(Hepatocyte growth factor,HGF),又称离散因子(scatter factor,SF),是目前发现的具有最强活性的刺激肝细胞再生的细胞因子,曾误以为它就是特异性肝细胞生长因子。但实际上,它还对多种细胞具有促有丝分裂、促移动、促形态发生的活性。HGF发挥其各种生物学作用,主要通过其受体c-Met实现,c-Met为具有酪氨酸蛋白激酶活性的膜受体,由原癌基因c-met编码,属于肝细胞生长因子受体家族中的一员。已有研究证实HGF在肿瘤细胞的侵袭和转移过程中起重要作用,而且在血管形成中也发挥重要作用。最近有研究显示,HGF还是淋巴管生成的促进因子。而血管、淋巴管的新生与肿瘤的侵袭和转移密切相关。研究显示,血管和淋巴管的形成是由内皮细胞的增殖、移行和成管等若干步骤组成的。但迄今,HGF在淋巴管形成过程中的作用及机制尚不清楚,HGF在人结直肠癌血管、淋巴管新生中的作用机制尚缺乏系统的研究。
血管内皮生长因子(VEGF)家族中的VEGF-A是肿瘤血管生成的重要因子;而VEGF-C和VEGF-D则是肿瘤淋巴管新生和淋巴结转移的重要调控因子。所以,探索肿瘤细胞如何调控VEGF家族的产生进而促进肿瘤细胞转移以及促进肿瘤血管、淋巴管生成就成为抗肿瘤脉管生成研究的重点问题。已有研究证实HGF可以上调结直肠癌VEGF-A的表达,但HGF能否调控结直肠癌细胞VEGF-C和VEGF-D的表达于国内外尚未见报道。
本实验采用形态与机能相结合的方法系统检测HGF对人脐静脉内皮细胞ECV-304,人真皮淋巴管内皮细胞(HDLEC),人结肠癌细胞SW480、SW620的增殖、移行行为以及HGF对SW480,SW620的VEGF-A,VEGF-C,VEGF-D表达的影响,以期为结直肠癌抗脉管生成寻找治疗新靶点。
本实验分两部分。
一HGF对人脐静脉内皮细胞ECV-304、人真皮淋巴管内皮细胞(HDLEC)的增殖、移行的影响
本实验采用MTT法分析HGF对细胞增殖的作用,流式细胞术检测HGF对细胞周期的影响,基底膜重建实验观察HGF对细胞移行的影响,透射电子显微镜观察加入HGF后细胞超微结构的变化。
结果显示:HGF能促进ECV-304、HDLEC增殖。HGF对ECV-304的促增殖作用呈质量浓度依赖关系。而对HDLEC的促增殖作用并不随着HGF浓度的增加而增强。流式细胞术结果显示,与对照组相比,ECV-304和HDLEC的HGF组中的G0 /G1期细胞均减少,S期细胞增多,G2/M期细胞增多。ECV-304增殖指数由对照组的37.8%增至47.5%,HDLEC增殖指数则由对照组的27.3%增至37.1%。HGF也能促进ECV-304、HDLEC移行。HGF≥10ng/ml时能有效促进ECV-304移行并且呈质量浓度依赖关系;HGF能有效促进HDLEC移行且呈质量浓度依赖关系。透射电子显微镜观察ECV-304和HDLEC细胞的超微结构发现,加入HGF后,两种细胞内的微丝均增多。
二HGF对人结肠癌细胞SW480和SW620的增殖、移行及VEGF-A、VEGF-C、VEGF-D表达水平的影响
本研究采用MTT法分析HGF对细胞增殖的作用。流式细胞术检测HGF对细胞周期的影响。基底膜重建实验观察HGF对细胞移行的影响。透射电子显微镜观察加入HGF后细胞超微结构的变化。应用Western Blotting检测两种细胞有无c-Met的表达以及加入HGF前后VEGF-A、VEGF-C、VEGF-D表达水平的变化。
结果显示,HGF能促进SW480和SW620增殖。HGF≥10ng/ml时开始促进SW480和SW620增殖,随着HGF浓度的增高SW620细胞增殖缓慢,但对SW480增殖作用不随着HGF浓度的增加而增强。流式细胞术结果显示,与对照组相比,SW480的HGF组中的G0 /G1期细胞减少,S期细胞增多,G2/M期细胞增多,但HGF对SW620细胞周期的影响不明显。SW480增殖指数由对照组的42.3%增至46.9%,SW620增殖指数由对照组的32.0%增至33.8%。HGF也能促进SW480和SW620移行,基底膜重建实验显示,HGF能明显促进SW480、SW620移行且呈质量浓度依赖关系。透射电子显微镜观察SW480和SW620细胞的超微结构发现,加入HGF后,两种细胞内的微丝均增多。Western Blotting结果显示,HGF的特异性受体c-Met表达于SW480和SW620细胞,加入HGF上调了SW480和SW620细胞的VEGF-A、VEGF-C、VEGF-D的表达水平。
结论
1 HGF可以促进人脐静脉内皮细胞ECV-304和人真皮淋巴管内皮细胞(HDLEC)的增殖和移行。HGF对二种细胞的促移行作用相似,均呈质量浓度依赖性,而促增殖作用有所不同。
2 HGF可以促进人结肠癌细胞SW480、SW620的增殖和移行作用。HGF对二种细胞的促移行作用相似,均呈质量浓度依赖性,而促增殖作用有所不同。
3 HGF可以上调SW480和SW620细胞的VEGF-A、VEGF-C、VEGF-D的表达水平。
Colorectal carcinoma is the second common malignant tumor in digestive tract. The main reason for its treatment failure is the development of hematogenous metastasis and lymphatic metastasis. It is shown that both the invasive capability of cancerous cells and the neogenesis of blood vessels and lympgatic vessels are closely related with the metastasis of colorectal carcinoma. And the density of blood vessels and lympgatic vessels is correctly related with lymphatic metastasis and the prognosis of colorectal cancer. Inhibiting the neogenesis of blood vessels can decrease the blood supply of the tumor, induce the apoptosis of cancerous cells and reduce the incidence of metastasis. The effective treatment target, especially hepatocyte growth factor (HGF), for colorectal cancer has been widely studied.
HGF, also known as scatter factor (SF), was first identified as a mediator of liver generation, but it is also a growth factor with pleiotropic effects including mitogen, mobility factor and morphogen and so on. HGF’s activities are mediated by a membrane-spanning receptor tyrosine kinase, the HGF receptor (HGF-R, also known as MET/c-met). It has been proved that HGF plays an important role in the infiltration and metastasis of cancerous cells and the neogenesis of blood vessels. Recently, many studies indicate that HGF may be an enhancing factor in the neogenesis of lymphatic vessels. Moreover, angiogenesis and lymphangiogenesis are directly related with the infiltration and metastasis of carcinoma. The angiogenesis and lymphangiogenesis is a process of proliferation, migration and canaliculization of the endothelial cells. However, the role of HGF in the the neogenesis of lympgatic vessels it is still unclear and it is short of systematic study of the role of HGF in the angiogenesis and lymphangiogenesis of colorectal carcinoma.
Vascular endothelial growth factor(VEGF)-A have been identified as one of the specific angiogenesis factors, while vascular endothelial growth factor (VEGF)-C and -D play important roles in lymphangiogenesis and metastasis. Therefore, to explore how the cancer cells control the birth of VEGF and then promote the metastasis of the cancer cells while inducing the angiogenesis and lymphangiogenesis becomes a key issue in the research. It has been proved that HGF can increase the BEGF-A expression of colorectal carcinoma, but whether it can adjust the expression of VEGF family has not been systematically studied.
This experiment uses a combinative method of form and function to examine systematically the effects of HGF on the proliferation and migration of ECV-304, HDLEC, SW480 and SW620 and the influence of HGF on the expression of FEGF-D in SW480 and SW620, in the hope of finding a new therapeutic target for restraining the neogenesis of blood vessels and lymphatic vessels of colorectal carcinoma.
The present study was devided into two parts.
1 The effects of HGF on the proliferation and migration of ECV-304 and human dermic lympgatic vessel endothelial cells (HDLEC).
The effect of HGF on the proliferation of endothelial cells was examined by MTT and the method Flow Cytometry (FCM) was used to test the effect of HGF on the cell cycle of endothelial cells. And the ultrastucture of endothelial cells treated with HGF was shown by the transmission electron microscope (TEM).HGF could promote the proliferation of ECV-304 in a concentration-dependent manner. But the effect of HGF on the proliferation of HDLEC didn’t increase with the concentration of HGF. The results of FCM showed that the cells of HGF group in G0 /G1 phase decreased obviously, but the cells in S phase and G2/M phase increased. The proliferation index of ECV-304 increased compared with the control group ( 47.5% vs 37.8%) and the proliferation index of HDLEC increased compared with the control group (37.1% vs 27.3%), which suggested that HGF mainly induced the cells to enter M phase. HGF could promote the migration of ECV-304 and HDLEC. HGF (>5ng/ml) obviously promoted the migration of ECV-304 in a concentration-dependent manner. Similarly, HGF effectively promoted the migration of HDLEC in a concentration-dependent manner. As shown by TEM, the microfilament of ECV-304 and HDLEC after being treated with HGF increased remarkably.
2 The effects of HGF on the proliferation and migration of SW480 and SW620 and on the expressions of VEGF-A, VEGF-C and VEGF-D.
MTT was used to analyse the effect of HGF on the proliferation and the effect of HGF on the cell cycle was determined by FCM. The effect of HGF on the migration of cells was studied by restoration of basal membrane; the ultrastructure of SW480 and SW620 after being treated with HGF was observed by TEM; the expressions of VEGF-A, VEGF-C and VEGF-D were determined by Western Blotting.
HGF (≥10ng/ml) could promoted the proliferation of SW480 and SW620. It promoted the proliferation of SW620 in a concentration-dependent manner but in a very feeble way. The effect of HGF on the proliferation of SW480 didn’t increase with the concentration of HGF. The results of FCM showed that the SW480 cells of HGF group in G0 /G1 phase decreased, but the cells in S phase and G2/M phase increased compared with the control group. However, there are weak changes in the SW620 cells of HGF group. The proliferation index of SW480 increased compared with that of the control group (46.9% vs 42.3%), and the proliferation index of SW620 increased compared with that of control group (33.8% vs 32.0%). HGF could promote the migration of SW480 and SW620. HGF promoted the migration of SW480 and SW620 in a concentration-dependent manner. As shown by TEM, the microfilaments of SW480 and SW620 increased remarkably after being treated with HGF. HGF upregulated the expressions of VEGF-A, VEGF-C and VEGF-D of SW480 and SW620 repectively.
Conclusion
1 HGF can promote the proliferation and migration of ECV-304 and HDLEC, and HGF can promote the migration of ECV-304 and HDLEC in a concentration-dependent manner.
2 HGF can promote the proliferation and migration of SW480 and SW620, and HGF can promote the migration of SW480 and SW620 in a concentration-dependent way.
3 HGF can promote the expressions of VEGF-A, VEGF-C and VEGF-D of SW480 and SW620 repectively.
引文
1 Park MK, Kim DK, Lee HJ. Adenoviral mediated hepatocyte growth factor gene attenuates hyperglycemia and beta cell destruction in over diabetic mice. Exp Mol Med, 2003, 35(6): 494-500
2 Nakamura T. Strueture and function of hepatocyte growth factor. Progress in growth factor research, 1991, 3(l): 67-85
3 Michalopoulos GK, DeFranees MC. Liver regeneration. Science, 1997, 276(5309): 60-66
4 Bottaro DP, Rubin JS, Faletto DL, et al. Identification of the hepatoeyte growth factor receptor as the c-met Prot- oncogene product. Science, 1991, 251(4995): 802-804
5 Park M, Dean M, CooPer CS, et al. Mechanism of met oncogene activation. Cell, 1986, 45(6): 895-904
6 Bussolino F, Di Renzo MF, Ziehe M, et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. the Journal of cell biology, 1992, 119(3): 629-641
7 Maeshima A, Zhang YQ, Furukawa M, Hepatocyte growth factor induces branching tubulogenesis in MDCK cells by modulating the activin-follistatin system. Kidney international, 2000, 58(4): 1511-1522
8 Matsumoto K, Nakamura T. Hepatocyte growth factor. molecular structure, roles in liver regeneration, and other biologieal functions. Critical reviews in oncogenesis, 1992,3(l-2): 27-54
9 Di Renzo MF, Olivero M, Giacomini A, et al. Over expression and amplification of the met/HGF receptor gene during the progression of colorectal cancer. Clin Cancer Res, 1995, 1(2): 147-154
10 Miyata Y, Ashida S, Nakamura T, et al. Over expression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo. Biochemical and biophysical research communications, 2003, 302(4): 892-897
11 陈珊, 金伟, 闵平等. 血管内皮生长因子家族及其受体与肿瘤血管生成研究进展. 生命科学, 2004,16(1): 19-23
12 Makinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transducer growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J, 2001, 20: 4762-73
13 Stacker SA, Caesar C, Baldwin ME, et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med, 2001, 7: 186-91
14 Wen G. Jianga, Tracey AMartin, Christian Parr, et al. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Critical Reviews in Oncology/Hematology , 2005, 53: 35-69
15 Kajiya K, Hirakawa S, Ma B, et al. growth factor promotes lymphatic vessel formation and function. EMBO J, 2005, 24: 2885-95
16 Cao R, Bjorndahl MA, Gallego MI, et al. Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action. Blood, 2006, 107: 3531-6
17 Bussolino F, Di Renzo MF, Ziche M, et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol, 1992, 119: 629-641
18 Grant DS, Kleinman HK, Goldberg ID, et al. Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci USA, 1993, 90: 1937-1941
19 Hai-Feng Duan, Chu-Tse Wu, Ying Lu, et al. Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells. Experimental Cell Research, 2004, 298: 593-601
20 Nakamura Y, Morishita R, Higaki J, et al. Hepatocyte growth factor is a novel member of the endothelium-specific growth factors: additive stimulatory effect of hepatocyte growth factor with basic fibroblast growth factor but not with vascular endothelial growth factor. Hypertens, 1996, 14: 1067-1072
21 Eirik Sundlisaeter, Aly Dicko, Per Oytein, et al. Lymphangiogenesis in colorectal cancer—Prognostic and therapeutic aspects. Int. J. Cancer, 2007, 121: 1401–1409
22 Goldman J, Rutkowski JM, Shields JD, et al. Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis. FASEB J, 2007, 21: 1-10
23 Bray D. Cell Movement. 2nd ed. NewYork: Garland Publishing, 2001: 47-79
24 Plastino J, Sykes C. The actin sling shot. Curr Opin Cell Biol, 2005, 17(1): 62-66
1 Gonzalez FJ, Quesada AR, Sevilla I, et al. Prognostic value of serum angiogenic activity in colorectal cancer patients. J Cell Mol Med, 2007, 11(1): 120-8
2 Matsumoto K, Nakayama Y, Inoue Y, et al. Lymphatic Microvessel Density is an Independent Prognostic Factor in Colorectal Cancer. Dis Colon Rectum, 2007, 50(3): 308-314
3 Wagner H. Multitarget therapy-the future of treatment for more than just functional dyspepsia. Phytomedicine, 2006, 13 Suppl 5: 122-9
4 Park MK, Kim DK, Lee HJ. Adenoviral mediated hepatocyte growth factor gene attenuates hyperglycemia and beta cell destruction in overt diabetic mice. Exp Mol Med, 2003, 35(6): 494-500
5 李宏武, 单吉贤. 人大肠癌组织肝细胞生长因子及其受体 c-met 的表达. 世界华人消化杂志, 2004, 12(9): 2199- 2201
6 Fukuura T, Miki C, Inoue T, et al. Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma. Br J Cancer, 1998, 78: 454-9
7 Britta S, Kurt S. Differences in the migration capacity of primary human colon carcinoma cells (SW480) and their lymph node metastatic derivatives (SW620). Cancer Letters, 1998, 131(1):55-64
8 Bray D. Cell Movement. 2nd ed. NewYork: GarlandPublishing, 2001: 47-79
9 Maruyama Y, Ono M, Kawahara A, et al. Tumor growthsuppression in pancreatic cancer by a putative metastasis suppressor gene Cap43 /NDRG1 /Drg-1 through modulation of angiogenesis. Cancer Res, 2006, 66(12): 6233-6242
10 Albig AR, Neil JR, Schiemann WP. Fibulins 3 and 5 antagonize tumor angiogenesis in vivo. Cancer Res, 2006, 66(5): 2621- 2629
11 Leung DW, Cachianes G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989, 246(4935): 1306-1309
12 Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular Permeability factor that promotes accumulation of ascites fluid. Science, 1983, 219(4587): 983-985
13 Bjorndahl MA, Cao R, Burton JB, et al. Vascular endothelial growth factor-a promotes peritumoral lymphangiogenesis and lymphatic metastasis. Cancer Res, 2005, 65: 9261-8
14 Schoppmann SF, Birner P, Stockl J, et al. Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol, 2002, 161: 947-56
15 Cao R, Bjorndahl MA, Gallego MI, et al. Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action. Blood, 2006, 107: 3531-6
1 梁后杰, 李建军. 重视恶性肿瘤淋巴管生成的研究. 第三军医大学学报, 2004, 26(24): 2179-2181
2 Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA, 1995, 92: 3566-70
3 Breiteneder-Geleff S, Soleiman A, Kowalski H, et al. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol, 1999, 154: 385-94
4 Banerji S, Ni J, Wang SX, et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol, 1999, 144: 789-801
5 Wigle JT, Oliver G. Prox1 function is required for the development of the murine lymphatic system. Cell, 1999, 98: 769-78
6 Makinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transducer growth, survival andmigratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J, 2001, 20: 4762-73
7 Partanen TA, Arola J, Saaristo A, et al.VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J, 2000, 14: 2087-96
8 Witmer AN, van Blijswijk BC, Dai J, et al. VEGFR-3 in adult angiogenesis. J Pathol, 2001, 195: 490-7
9 Kubo H, Fujiwara T, Jussila L, et al. Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood, 2000, 96: 546-53
10 Stacker SA, Achen MG, Jussila L, et al. Lymphangiogenesis and cancer metastasis. Nat Rev Cancer, 2002, 2: 573-583
11 Schacht V, Ramirez MI, Hong YK, et al. T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J, 2003, 22: 3546-56
12 Breiteneder Geleff S, Soleiman A, Kowalski H, et al. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: Podoplanin as a specific marker for lymphatic endothelium. Am J Pathal, 1999, 154 (2): 385-394
13 Baneji S, Ni J, Wang SX, et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific recepter for hyaluronan. J Cell Biol, 1999, 144 (4): 789-801
14 Mouta Carreira C, Nasser SM, di Tomaso E, et al. LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis. Cancer Res, 2001, 61: 8079-84
15 Jackson DG. Biology of the lymphatic marker LYVE-1 and applications in research into lymphatic trafficking and lymphangiogenesis. APMIS, 2004, 112: 526-38
16 Wigle J T, Harvey N, Detmar M, et al. An essential role for Prox-1 in the induction of the lymphatic endothelial cell phenotype. EMBO J, 2002, 21: 1505-1513
17 Kazama S, Kitayama J, Watanabe T, et al. Expression pattern of vascular endothelial growth factor-C in human colorectal normal mucosa and neoplastic mucosa. Hepatogastroenterology, 2004, 51: 91-5
18 Skobe M, Hawighorst T, Jackson DG, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med, 2001, 7: 192-8
19 Onogawa S, Kitadai Y, Tanaka S, et al. Expression of VEGF-C and VEGF-D at the invasive edge correlates with lymph node metastasis and prognosis of patients with colorectal carcinoma. Cancer Sci, 2004, 95: 32-9
20 Kawakami M, Furuhata T, Kimura Y, et al. Quantification of vascular endothelial growth factor-C and its receptor-3 messenger RNA with real-time quantitative polymerase chain reaction as a predictor of lymph node metastasis in human colorectal cancer. Surgery, 2003, 133: 300-8
21 Kawakami M, Furuhata T, Kimura Y, et al. Expression analysis of vascular endothelial growth factors and their relationships to lymph node metastasis in human colorectal cancer. J Exp Clin Cancer Res, 2003, 22: 229-37
22 Jia YT, Li ZX, He YT, et al. Expression of vascular endothelial growth factor-C and the relationship between lymphangiogenesis and lymphatic metastasis in colorectal cancer. World J Gastroenterol, 2004, 10: 3261-3
23 Mattila MM, Ruohola JK, Karpanen T, et al. VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int J Cancer, 2002, 98: 946-51
24 Hirakawa S, Brown LF, Kodama S, et al. VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood, 2006, 109: 1010-17
25 Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J, 1996, 15: 290-98
26 Makinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transducer growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J, 2001, 20: 4762-73
27 Stacker SA, Caesar C, Baldwin ME, et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med, 2001, 7: 186-91
28 Joukov V, Sorsa T, Kumar V, et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J, 1997, 16: 3898-911
29 Stacker SA, Stenvers K, Caesar C, et al. Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem, 1999, 274: 32127-36
30 Funaki H, Nishimura G, Harada S, et al. Expression of vascular endothelial growth factor D is associated with lymph node metastasis in human colorectal carcinoma. Oncology, 2003, 64: 416-22
31 White JD, Hewett PW, Kosuge D, et al. Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma. Cancer Res, 2002, 62: 1669-75
32 George ML, Tutton MG, Janssen F, et al. VEGF-A, VEGF-C, and VEGF-D in colorectal cancer progression. Neoplasia, 2001, 3: 420-7
33 Hanrahan V, Currie MJ, Gunningham SP, et al. The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma-carcinoma sequence during colorectal cancer progression. J Pathol, 2003, 200: 183-94
34 Saad RS, Kordunsky L, Liu YL, et al. Lymphatic microvessel density as prognostic marker in colorectal cancer. Mod Pathol, 2006, 19: 1317-23
35 Bjorndahl MA, Cao R, Burton JB, et al. Vascular endothelial growth factor-a promotes peritumoral lymphangiogenesis and lymphatic metastasis. Cancer Res, 2005, 65: 9261-8
36 Hirakawa S, Kodama S, Kunstfeld R, et al. VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J ExpMed, 2005, 201: 1089-99
37 Schoppmann SF, Birner P, Stockl J, et al. Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol, 2002, 161: 947-56
38 Bikfalvi A, Klein S, Pintucci G, et al. Biological roles of fibroblast growth factor-2. Endocr Rev, 1997, 18: 26-45
39 Chang LK, Garcia-Cardena G, Farnebo F, et al. Dosedependent response of FGF-2 for lymphangiogenesis. Proc Natl Acad Sci USA, 2004, 101: 11658-63
40 Elagoz S, Egilmez R, Koyuncu A, et al. The intratumoral microvessel density and expression of bFGF and nm23-H1 in colorectal cancer. Pathol Oncol Res, 2006, 12: 21-7
41 Cao R, Bjorndahl MA, Religa P, et al. PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell, 2004, 6: 333-45
42 Tammela T, Saaristo A, Lohela M, et al. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood, 2005, 105: 4642-8
43 Morisada T, Oike Y, Yamada Y,et al. Angiopoietin-1promotes LYVE-1-positive lymphatic vessel formation. Blood, 2005, 105: 4649-56
44 Gale NW, Thurston G, Hackett SF,et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell, 2002, 3: 411-23
45 Kajiya K, Hirakawa S, Ma B, et al. growth factor promotes lymphatic vessel formation and function. EMBO J, 2005, 24: 2885-95.
46 Cao R, Bjorndahl MA, Gallego MI, et al. Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action. Blood, 2006, 107: 3531-6.
47 Goldman J, Rutkowski JM, Shields JD, et al. Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis. FASEB J, 2007, 21: 1-10
48 Fukuura T, Miki C, Inoue T, et al. Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma. Br J Cancer, 1998, 78: 454-9
49 Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971, 285: 1182-6
50 Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med, 2006, 355: 2542-50
51 Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science, 2005, 307: 58-62
52 Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med, 2004, 10: 145-7
53 Karpanen T, Egeblad M, Karkkainen MJ, et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res, 2001, 61: 1786-90
54 Kane RC, Farrell AT, Saber H, et al. Sorafenib for the treatment of advanced renal cell carcinoma. Clin Cancer Res, 2006, 12: 7271-78
55 News in brief. Nat Rev Drug Discov, 2005, 4: 448-49
56 Rebhun RB, Langley RR, Yokoi K, et al. Targeting receptor tyrosine kinase on lymphatic endothelial cells for the therapy of colon cancer lymph node metastasis. Neoplasia, 2006, 8: 747-57
57 Busby JE, Kim SJ, Yazici S, et al. Therapy of multidrug resistant human prostate tumors in the prostate of nude mice by simultaneous targeting of the epidermal growth factor receptor and vascular endothelial growth factor receptor on tumor-associated endothelial cells. Prostate, 2006, 66: 1788-98