肝癌细胞系中VEGFR-1的表达及其功能的初步研究
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摘要
[目的]
血管内皮生长因子(VEGF)及其受体在参与包括肿瘤血管在内的病理性血管形成中的作用已被广泛研究和认识。近年来,随着研究的深入,VEGFR-1在肿瘤侵袭进展中的非促血管形成功能越来越受到重视。结肠癌、胰腺癌、乳腺癌的体内外研究中显示,肿瘤组织及体外细胞系均存在有功能性VEGFR-1,其激活后可以通过诱导肿瘤细胞发生上皮细胞向间质细胞转化(EMT)以及促进MMPs的分泌及活性功能增强两方面来增加肿瘤细胞的侵袭转移能力。原发性肝细胞癌(HCC)是VEGF高表达的富血供、高侵袭性实体肿瘤。国内外对HCC中VEGFR-1的表达情况及其功能和机制尚没有系统研究。
本研究检测了国内几种常见肝癌细胞系VEGFR-1的表达情况,并在SMMC-7721肝癌细胞系上,用VEGF-B167特异性激活VEGFR-1,检测肿瘤细胞核转录因子snail的表达变化及MMP-9表达和活性功能变化。同时检测不同侵袭能力肝癌细胞系：MHCC-97H、SMMC-7721、HepG2及正常胎肝细胞系LO2中Snail的表达水平,结合文献,对肝癌细胞系中VEGFR-1的功能和机制做初步研究探讨,为寻找防治肝癌侵袭转移的靶点提供新的理论依据。
[方法]
培养细胞系：LO2、HepG2、SMMC-7721、MHCC-97H, Western blot(WB)方法检测VEGFR-1、snail蛋白的表达,凝胶成像系统扫描各条带灰度值对各组进行半定量分析比较；SMMC-7721细胞系换无胎牛血清1640培养基,饥饿过夜,次日加入VEGF-B167 (50ng/ml)处理,同时设置非特异性IgG对照组和完全空白对照,WB法检测各种处理组snail蛋白的表达和VEGF-B167刺激后不同时间点(0h、8h、24h、28h、32h) snail的表达水平变化；用明胶酶谱法检测VEGF-B167刺激SMMC-7721细胞系培养上清中MMP-9活性功能的表达。进行统计分析。
[结果]
1、四种细胞系中均有VEGFR-1的存在,半定量灰度分析比较结果显示：MHCC-97H表达最高,SMMC-7721次之,而正常胎肝细胞系LO2也有较低的表达。
2、三种肝癌细胞系中均有Snail蛋白的表达,半定量灰度分析结果显示；MHCC-97H的表达最高,SMMC-7721次之,HepG2表达较低,正常胎肝细胞LO2未见表达。
3、VEGF-B167激活SMMC-7721肝癌细胞系VEGFR-1后,Snail蛋白表达逐渐增加,并在28h点达到最高,32h点下降。
实验组
非特异性IgG对照组
4、VEGF-B167激活SMMC-7721细胞系VEGFR-1,28h点与非特异性IgG组、完全空白对照组相比,实验组Snail表达增高。
5、明胶酶谱检测VEGF-B167激活SMMC-7721细胞系VEGFR-1后,28h点与非特异性IgG组、完全空白对照组相比实验组MMP-9的活性功能增强,未有明显的MMP-2 (68KD)位置负染条带的显示。
6、WB检测VEGF-B167激活SMMC-7721细胞系VEGFR-1后,不同时间点MMP-9表达水平的变化。24h点出现条带,随着时间的推移,MMP-9的表达有所增高,48h条带消失。
[结论]
VEGFR-1表达在肝癌细胞系,其激活后引起以MMP-9表达增高和活性功能增强为表现的肝癌细胞侵袭能力的增强,而Snail基因参与介导了此过程。
【Objective】
The role of vascular endothelial growth factor(VEGF) and its receptors participating in the pathological blood vessel formation has been widely known.In recent years,with the deep research,people has paid more attention to the non-angiogenesis-promoting fuction of VEGFR-1 on tumor invasion and progession.The in vivo or in vitro study of colon cancer,pancreatic carcinoma,breast cancer has shown that tumor tissues and cell lines both express functional VEGFR-1,and the activation of VEGFR-1 can inhance tumor invasion and metastasis by inducing the epithelial cells change to the stroma cells(EMT) and promoting the secretion and active function of MMPs. Hepatocellular carcinoma(HCC) is the solid tumor rich in the expression of VEGF、blood vessel and invasion.Up to now,there is no systematic study about the expression、function and mechanism of VEGFR-1 in HCC at home and abroad.
In this study,we detected the expression of VEGFR-1 protein in different hepatocarcinoma cell lines,we also detected the expression of nuclear transcription factor:Snail protein and the functional changes of MMP-9 in cell culture supernatant which were both stimulated by VEGF-B167 from SMMC-7721 cell line;at the same time we detected the expression of Snail protein in different invasive cell lines:MHCC-97H、SMMC-7721、HepG2 and the normal liver cell line:LO2. To make the preliminary research of the function and mechanism of VEGFR-1 in hepatocellular carcinoma cells,and to provide a new theoretical basis on the prevention and treatment of hepatocellular carcinoma in future.
[Methods]
Culture various cell lines:LO2、HepG2、SMMC-7721、MHCC-97H.To detect VEGFR-1、Snai protein expression by Western Blot (WB),.SMMC-7721 cell line were renewed by non-FBS 1640 medium,starvation overnight,the next day by adding VEGF-B167,at the same time set the IgG isotype control and blank control, to detect the Snail protein expression of different treatment groups and Snail protein expression changes stimulated by VEGF-B167 at different time points(0h、8h、24h、28h、32h) by WB; to detect the functional activity of MMP-9 in SMMC-7721 cell line supernant treated by VEGF-B167、IgG isotype control and blank control through gelatin zymography. At last for statistical analysis.
[Results]
1、VEGFR-1 protein expressed in LO2、HepG2、SMMC-7721、MHCC-97H cell lines,semiquantitative gray scale analysis show that the highest expression of VEGFR-1 in MHCC-97H cell,the moderate in SMMC-7721, while the lowest in LO2.
2、MHCC-97H、SMMC-7721、HepG2 hepatoma cell lines expressed Snail protein and LO2 did not expression Snail. semiquantitative gray scale analysis show that the highest expression of Snail in MHCC-97H cell,the moderate in SMCC-7721, while the lowest in HepG2.
3、At 28h the protein expression of Snail is to the peak by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721cell line,while at 32h the snail expression gradually dreased. VEGF-B167 stimulation group: IgG isotype contol
4, The expression of Snail was significantly increased by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721 cell line at 28h as to IgG isotype control、the blank group.
5、The active function of MMP-9 was significantly increased by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721 cell line at 28h as to IgG isotype control、the blank group by gelatin-zymogram.The MMP-2 band (64KD) is unnoticeable.
6、The protein expression of MMP-9 in SMMC-7721 cell line at the different time points(0h、24h、28h、32h、48h).24h the band appeared,with time,the expression of MMP-9 gradually inceased,48h the band disappeared.
[Conclusions]
VEGFR-l expressed in hepatoma cell lines and its activation can inhance the invasive activity of cell lines through the increased expression and enhanced function of MMP-9,while Snail participated and mediated in this process.
血管内皮生长因子(VEGF)及其受体在参与包括肿瘤血管在内的病理性血管形成中的作用已被广泛研究和认识。近年来,随着研究的深入,VEGFR-1在肿瘤侵袭进展中的非促血管形成功能越来越受到重视。结肠癌、胰腺癌、乳腺癌的体内外研究中显示,肿瘤组织及体外细胞系均存在有功能性VEGFR-1,其激活后可以通过诱导肿瘤细胞发生上皮细胞向间质细胞转化(EMT)以及促进MMPs的分泌及活性功能增强两方面来增加肿瘤细胞的侵袭转移能力。原发性肝细胞癌(HCC)是VEGF高表达的富血供、高侵袭性实体肿瘤。国内外对HCC中VEGFR-1的表达情况及其功能和机制尚没有系统研究。
本研究检测了国内几种常见肝癌细胞系VEGFR-1的表达情况,并在SMMC-7721肝癌细胞系上,用VEGF-B167特异性激活VEGFR-1,检测肿瘤细胞核转录因子snail的表达变化及MMP-9表达和活性功能变化。同时检测不同侵袭能力肝癌细胞系：MHCC-97H、SMMC-7721、HepG2及正常胎肝细胞系LO2中Snail的表达水平,结合文献,对肝癌细胞系中VEGFR-1的功能和机制做初步研究探讨,为寻找防治肝癌侵袭转移的靶点提供新的理论依据。
[方法]
培养细胞系：LO2、HepG2、SMMC-7721、MHCC-97H, Western blot(WB)方法检测VEGFR-1、snail蛋白的表达,凝胶成像系统扫描各条带灰度值对各组进行半定量分析比较；SMMC-7721细胞系换无胎牛血清1640培养基,饥饿过夜,次日加入VEGF-B167 (50ng/ml)处理,同时设置非特异性IgG对照组和完全空白对照,WB法检测各种处理组snail蛋白的表达和VEGF-B167刺激后不同时间点(0h、8h、24h、28h、32h) snail的表达水平变化；用明胶酶谱法检测VEGF-B167刺激SMMC-7721细胞系培养上清中MMP-9活性功能的表达。进行统计分析。
[结果]
1、四种细胞系中均有VEGFR-1的存在,半定量灰度分析比较结果显示：MHCC-97H表达最高,SMMC-7721次之,而正常胎肝细胞系LO2也有较低的表达。
2、三种肝癌细胞系中均有Snail蛋白的表达,半定量灰度分析结果显示；MHCC-97H的表达最高,SMMC-7721次之,HepG2表达较低,正常胎肝细胞LO2未见表达。
3、VEGF-B167激活SMMC-7721肝癌细胞系VEGFR-1后,Snail蛋白表达逐渐增加,并在28h点达到最高,32h点下降。
实验组
非特异性IgG对照组
4、VEGF-B167激活SMMC-7721细胞系VEGFR-1,28h点与非特异性IgG组、完全空白对照组相比,实验组Snail表达增高。
5、明胶酶谱检测VEGF-B167激活SMMC-7721细胞系VEGFR-1后,28h点与非特异性IgG组、完全空白对照组相比实验组MMP-9的活性功能增强,未有明显的MMP-2 (68KD)位置负染条带的显示。
6、WB检测VEGF-B167激活SMMC-7721细胞系VEGFR-1后,不同时间点MMP-9表达水平的变化。24h点出现条带,随着时间的推移,MMP-9的表达有所增高,48h条带消失。
[结论]
VEGFR-1表达在肝癌细胞系,其激活后引起以MMP-9表达增高和活性功能增强为表现的肝癌细胞侵袭能力的增强,而Snail基因参与介导了此过程。
【Objective】
The role of vascular endothelial growth factor(VEGF) and its receptors participating in the pathological blood vessel formation has been widely known.In recent years,with the deep research,people has paid more attention to the non-angiogenesis-promoting fuction of VEGFR-1 on tumor invasion and progession.The in vivo or in vitro study of colon cancer,pancreatic carcinoma,breast cancer has shown that tumor tissues and cell lines both express functional VEGFR-1,and the activation of VEGFR-1 can inhance tumor invasion and metastasis by inducing the epithelial cells change to the stroma cells(EMT) and promoting the secretion and active function of MMPs. Hepatocellular carcinoma(HCC) is the solid tumor rich in the expression of VEGF、blood vessel and invasion.Up to now,there is no systematic study about the expression、function and mechanism of VEGFR-1 in HCC at home and abroad.
In this study,we detected the expression of VEGFR-1 protein in different hepatocarcinoma cell lines,we also detected the expression of nuclear transcription factor:Snail protein and the functional changes of MMP-9 in cell culture supernatant which were both stimulated by VEGF-B167 from SMMC-7721 cell line;at the same time we detected the expression of Snail protein in different invasive cell lines:MHCC-97H、SMMC-7721、HepG2 and the normal liver cell line:LO2. To make the preliminary research of the function and mechanism of VEGFR-1 in hepatocellular carcinoma cells,and to provide a new theoretical basis on the prevention and treatment of hepatocellular carcinoma in future.
[Methods]
Culture various cell lines:LO2、HepG2、SMMC-7721、MHCC-97H.To detect VEGFR-1、Snai protein expression by Western Blot (WB),.SMMC-7721 cell line were renewed by non-FBS 1640 medium,starvation overnight,the next day by adding VEGF-B167,at the same time set the IgG isotype control and blank control, to detect the Snail protein expression of different treatment groups and Snail protein expression changes stimulated by VEGF-B167 at different time points(0h、8h、24h、28h、32h) by WB; to detect the functional activity of MMP-9 in SMMC-7721 cell line supernant treated by VEGF-B167、IgG isotype control and blank control through gelatin zymography. At last for statistical analysis.
[Results]
1、VEGFR-1 protein expressed in LO2、HepG2、SMMC-7721、MHCC-97H cell lines,semiquantitative gray scale analysis show that the highest expression of VEGFR-1 in MHCC-97H cell,the moderate in SMMC-7721, while the lowest in LO2.
2、MHCC-97H、SMMC-7721、HepG2 hepatoma cell lines expressed Snail protein and LO2 did not expression Snail. semiquantitative gray scale analysis show that the highest expression of Snail in MHCC-97H cell,the moderate in SMCC-7721, while the lowest in HepG2.
3、At 28h the protein expression of Snail is to the peak by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721cell line,while at 32h the snail expression gradually dreased. VEGF-B167 stimulation group: IgG isotype contol
4, The expression of Snail was significantly increased by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721 cell line at 28h as to IgG isotype control、the blank group.
5、The active function of MMP-9 was significantly increased by the stimulation of VEGF-B167 to VEGFR-1 in SMMC-7721 cell line at 28h as to IgG isotype control、the blank group by gelatin-zymogram.The MMP-2 band (64KD) is unnoticeable.
6、The protein expression of MMP-9 in SMMC-7721 cell line at the different time points(0h、24h、28h、32h、48h).24h the band appeared,with time,the expression of MMP-9 gradually inceased,48h the band disappeared.
[Conclusions]
VEGFR-l expressed in hepatoma cell lines and its activation can inhance the invasive activity of cell lines through the increased expression and enhanced function of MMP-9,while Snail participated and mediated in this process.
引文
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50.Miyoshi, A., Kitajima, Y., Sumi, K., Sato, K., Hagiwara, A., Koga, Y. And Miyazaki, K. Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells. Br. J. Cancer2004; 90,1265-1273.
51.Yao, J., Xiong, S., Klos, K., Nguyen, N., Grijalva, R., Li, P. and Yu, D. Multiple signalling pathways involved in activation of matrix metalloproteinase-9 (MMP-9) by heregulin-betal in human breast cancer cells. Oncogene 2001;20, 8066-8074.
52. Grau, Y., Carteret, C., and Simpson, P. Mutations and chromosomal rearrangements affecting the expression of snail, a gene involved in embryonic patterning in Drosophila melanogaster. Genetics,1984,108:347-360.
53. Leptin, M., and Grunewald, B. Cell shape changes during gastrulation in Drosophila. Development (Camb.),1990,110:73-84.
54. Oda, H., Tsukita, S., and Takeichi, M. Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev. Biol.,1998; 203: 435-450.
55. Smith, D. E., Franco del Amo, F., and Gridley, T. Isolation of Sna, a mouse gene homologous to the Drosophila genes snail and escargot:its expression pattern suggests multiple roles during postimplantation development. Development (Camb.),1992; 116:1033-1039.
56. Guarino M.Epithelial-mesenchymal transition and tumour invasion.Int J Biochem Cell Biol,2007,39(12):2153-2160.
57. Keishi Sugimachi,Shinji Tanaka,Toshifumi Kameyama etal Transcriptional repressor Snail and progression of human hepatocellular carcinoma Clinical cancer res 2003;(9),2657-2664
1. Hicklin D, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.J Clin Oncol.2005;23:1011-1027.
2. Fischer C, Mazzone M, Jonckx B; Carmeliet P. FLT1 and its ligands VEGFB and PlGF:drug targets for anti-angiogenic therapy? Nat Review Cancer. 2008;8:942-956.
3. Wu Y, Zhong Z, Huber J, et al. Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer. Clin Cancer Res. 2006;12:6573-6584.
4. Decaussin M, Sartelet H, Robert C, et al. Expression of vascular endothelial growth factor (VEGF) and its 2 receptors (VEGF-R1-FLT1 and VEGF-R2-Flkl/Kdr) in non-small cell lung carcinomas (NSCLCS):correlation with angiogenesis and survival. J Pathol.1999; 188:369-377.
5. Sato K, Sasaki R, Ogura Y, et al. Expression of vascular endothelial growth factor gene and its receptor (flt-1) gene in urinary bladder cancer. Tohoku J Exp Med. 1998;185:173-184.
6. Wang ES, Teruya-Feldstein J, Wu Y, Zhu Z, Hicklin DJ,Moore MA. Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo. Blood.2004; 104:2893-2902.
7. Mylona E, Alexandrou P, Giannopoulou I, et al. The prognostic value of vascular endothelial growth factors (VEGFs)-A and -B and their receptor, VEGFR-1, in invasive breast carcinoma. Gynecol Oncol.2007;104:557-563.
8. Seto T, Higashiyama M, Funai H, et al. Prognostic value of expression of vascular endothelial growth factor and its flt-1 and KDR receptors in stage I non-small-cell lung cancer.Lung Cancer.2006;53:91-96.
9. Maru Y, Yamaguchi S, Shibuya M. Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials. Oncogene. 1998;16:2585-2595.
10. Bates RC, Goldsmith JD, Bachelder RE, et al. Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids. Curr Biol.2003;13:1721-1727.
11. Yang AD, Camp ER, Fan F, et al. Vascular endothelial growth factor receptor-1
activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells. Cancer Res.2006;66:46-51.
12. Fan F, Wey JS, McCarty MF, et al. Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene. 2005;24:2647-2653.
13. Price DJ, Miralem T, Jiang S, Steinberg R, Avraham H. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells. Cell Growth Differ.2001;12:129-135.
14. Wey JS, Fan F, Gray MJ, et al. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer. 2005; 104:427-438.
15. Wu Y, Hooper AT, Zhong Z, et al. The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma. Int J Cancer.2006;119:1519-1529.
16. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science.1992;255:989-991.
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19. Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci USA.1998;95:9349-9354.
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21. Lacal PM, Morea V, Ruffini F, et al. Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide. Euro J Cancer.2008;44:1914-1921.
22. Ponticelli S, Marasco D, Tarallo V, et al. Modulation of angiogenesis by a tetrameric tripeptide that antagonizes vascular endothelial growth factor receptor 1. J Biol Chem.2008;283:34250-34259.
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25. Autiero M, Waltenberger J, Communi D, et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Fltl and Flkl. Nat Med. 2003;9:936-943.
26. Wei SC, Tsao PN, Yu SC, et al. Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut.2005;54:666-672.
27. Parr C, Watkins G, Boulton M, Cai J, Jiang WG. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer. 2005;41:2819-2827.
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36. Decaussin M, Sartelet H, Robert C, et al. Expression of vascular endothelial growth factor (VEGF) and its 2 receptors (VEGF-R1-FLT1 and VEGF-R2-Flkl/Kdr) in non-small cell lung carcinomas (NSCLCS):correlation with angiogenesis and survival. J Pathol.1999;188:369-377.
37.Sato K, Sasaki R, Ogura Y, et al. Expression of vascular endothelial growth factor gene and its receptor (flt-1) gene in urinary bladder cancer. Tohoku J Exp Med. 1998;185:173-184.
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50.Miyoshi, A., Kitajima, Y., Sumi, K., Sato, K., Hagiwara, A., Koga, Y. And Miyazaki, K. Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells. Br. J. Cancer2004; 90,1265-1273.
51.Yao, J., Xiong, S., Klos, K., Nguyen, N., Grijalva, R., Li, P. and Yu, D. Multiple signalling pathways involved in activation of matrix metalloproteinase-9 (MMP-9) by heregulin-betal in human breast cancer cells. Oncogene 2001;20, 8066-8074.
52. Grau, Y., Carteret, C., and Simpson, P. Mutations and chromosomal rearrangements affecting the expression of snail, a gene involved in embryonic patterning in Drosophila melanogaster. Genetics,1984,108:347-360.
53. Leptin, M., and Grunewald, B. Cell shape changes during gastrulation in Drosophila. Development (Camb.),1990,110:73-84.
54. Oda, H., Tsukita, S., and Takeichi, M. Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev. Biol.,1998; 203: 435-450.
55. Smith, D. E., Franco del Amo, F., and Gridley, T. Isolation of Sna, a mouse gene homologous to the Drosophila genes snail and escargot:its expression pattern suggests multiple roles during postimplantation development. Development (Camb.),1992; 116:1033-1039.
56. Guarino M.Epithelial-mesenchymal transition and tumour invasion.Int J Biochem Cell Biol,2007,39(12):2153-2160.
57. Keishi Sugimachi,Shinji Tanaka,Toshifumi Kameyama etal Transcriptional repressor Snail and progression of human hepatocellular carcinoma Clinical cancer res 2003;(9),2657-2664
1. Hicklin D, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.J Clin Oncol.2005;23:1011-1027.
2. Fischer C, Mazzone M, Jonckx B; Carmeliet P. FLT1 and its ligands VEGFB and PlGF:drug targets for anti-angiogenic therapy? Nat Review Cancer. 2008;8:942-956.
3. Wu Y, Zhong Z, Huber J, et al. Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer. Clin Cancer Res. 2006;12:6573-6584.
4. Decaussin M, Sartelet H, Robert C, et al. Expression of vascular endothelial growth factor (VEGF) and its 2 receptors (VEGF-R1-FLT1 and VEGF-R2-Flkl/Kdr) in non-small cell lung carcinomas (NSCLCS):correlation with angiogenesis and survival. J Pathol.1999; 188:369-377.
5. Sato K, Sasaki R, Ogura Y, et al. Expression of vascular endothelial growth factor gene and its receptor (flt-1) gene in urinary bladder cancer. Tohoku J Exp Med. 1998;185:173-184.
6. Wang ES, Teruya-Feldstein J, Wu Y, Zhu Z, Hicklin DJ,Moore MA. Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo. Blood.2004; 104:2893-2902.
7. Mylona E, Alexandrou P, Giannopoulou I, et al. The prognostic value of vascular endothelial growth factors (VEGFs)-A and -B and their receptor, VEGFR-1, in invasive breast carcinoma. Gynecol Oncol.2007;104:557-563.
8. Seto T, Higashiyama M, Funai H, et al. Prognostic value of expression of vascular endothelial growth factor and its flt-1 and KDR receptors in stage I non-small-cell lung cancer.Lung Cancer.2006;53:91-96.
9. Maru Y, Yamaguchi S, Shibuya M. Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials. Oncogene. 1998;16:2585-2595.
10. Bates RC, Goldsmith JD, Bachelder RE, et al. Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids. Curr Biol.2003;13:1721-1727.
11. Yang AD, Camp ER, Fan F, et al. Vascular endothelial growth factor receptor-1
activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells. Cancer Res.2006;66:46-51.
12. Fan F, Wey JS, McCarty MF, et al. Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene. 2005;24:2647-2653.
13. Price DJ, Miralem T, Jiang S, Steinberg R, Avraham H. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells. Cell Growth Differ.2001;12:129-135.
14. Wey JS, Fan F, Gray MJ, et al. Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer. 2005; 104:427-438.
15. Wu Y, Hooper AT, Zhong Z, et al. The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma. Int J Cancer.2006;119:1519-1529.
16. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science.1992;255:989-991.
17. Sawano A, Takahashi T, Yamaguchi S, Aonuma M, Shibuya M. Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor. Cell Growth Differ. 1996;7:213-221.
18. Hiratsuka S, Maru Y, Okada A, Seiki M, Noda T, Shibuya M. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res.2001;61:1207-1213.
19. Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci USA.1998;95:9349-9354.
20. El-Mousawi M, Tchistiakova L, Yurchenko L, et al. A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library. J Biol Chem. 2003;278:46681-46691.
21. Lacal PM, Morea V, Ruffini F, et al. Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide. Euro J Cancer.2008;44:1914-1921.
22. Ponticelli S, Marasco D, Tarallo V, et al. Modulation of angiogenesis by a tetrameric tripeptide that antagonizes vascular endothelial growth factor receptor 1. J Biol Chem.2008;283:34250-34259.
23. Luttun A, Tjwa M, Moons L, et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med.2002;8:831-840.
24. Bae DG, Kim TD, Li G, Yoon WH, Chae CB. Anti-fltl peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis. Clin Cancer Res.2005; 11:2651-2661.
25. Autiero M, Waltenberger J, Communi D, et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Fltl and Flkl. Nat Med. 2003;9:936-943.
26. Wei SC, Tsao PN, Yu SC, et al. Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut.2005;54:666-672.
27. Parr C, Watkins G, Boulton M, Cai J, Jiang WG. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer. 2005;41:2819-2827.
28. Chen CN, Hsieh FJ, Cheng YM, et al. The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer. Cancer Lett. 2004;213:73-82.
29. Lacal PM, Failla CM, Pagani E, et al. Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor. J Invest Dermatol.2000;115:1000-1007.
30. Ho MC, Chen CN, Lee H, et al. Placenta growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma. Cancer Lett.2007;250:237-249.
31. Fischer C, Jonckx B, Mazzone M, et al. Anti-PIGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell. 2007;131:463-475.
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