摘要
研究背景和目的:骨桥蛋白(Osteopontin, OPN)是一种分泌型钙结合的磷酸化糖蛋白。在多种高转移性的恶性肿瘤细胞和患者的血清中均有OPN的过度表达。OPN诱导肿瘤细胞的浸润和转移与基质金属蛋白酶MMP-2和MMP-9的活化密切相关,OPN通过IKK (I kappa B kinase)酶依赖的信号通道诱导NF-κB (nuclear factor kappaB)介导的MMP-2和MMP-9的活化。本研究旨在探讨OPN在人前列腺癌PC-3细胞的增殖和侵袭过程中所发挥的作用,为IKK-1和IKK-2酶在NF-κB介导的信号通道中可能的功能提供实验依据。方法:构建四种含OPNshRNA的真核表达载体,分别转染人前列腺癌PC-3细胞,然后采用半定量RT-PCR方法筛选能显著抑制OPN基因表达的重组质粒。建立稳定转染OPNshRNA重组质粒的PC-3细胞株,并且应用不同浓度的IKK抑制剂Ⅶ分别抑制IKK-1和IKK-2两种酶的活性,然后采用Real-time PCR和Western blot分别检测OPN、MMP-2和MMP-9三种mRNA和蛋白的表达水平,ELISA法检测加入OPN蛋白培养的PC-3细胞的条件培养基中MMP-2、MMP-9的浓度。流式细胞术、MTT法和Transwell实验分别检测细胞生长周期变化、细胞增殖和和侵袭能力变化。应用成瘤实验观察OPN shRNA表达载体对PC-3细胞在裸鼠体内的生长潜能的影响。结果:RT-PCR方法成功筛选出能显著抑制前列腺癌PC-3细胞中OPN表达的OPN shRNA重组质粒PGPU6/GFP/Neo-OPN2,建立了稳定传代的细胞株PCs, PC/Vect和PC/OPN2。与PCs组相比,转染重组质粒的细胞PC/OPN2中OPN、MMP-2和MMP-9三种蛋白的表达量分别下降了55.22%、51.71%和28.35%(P<0.05),PC/OPN2细胞的增殖、迁移和侵袭能力受到明显抑制。此外,特异性地抑制IKK-2酶的活性能显著降低MMP-2和MMP-9的表达水平,而抑制IKK-1酶的活性对MMP-2和MMP-9的表达水平没有明显影响。结论:OPN shRNA表达载体介导的OPN基因沉默不仅能下调OPN的表达而且能抑制MMP-2和MMP-9的分泌,OPN shRNA对前列腺癌PC-3细胞的增殖、迁移和侵袭能力具有抑制作用,而且该过程与MMP-2和MMP-9两种酶的表达减少密切相关,此外,在NF-κB介导的OPN诱导MMP-2和MMP-9活化的过程中IKK-2酶的活性发挥了重要的作用。
Objective:Substantial data have linked osteopontin (OPN), a secreted phosphoglycoprotein, with tumor progression and metastatic spread. OPN regulates cell migration and invasion in a variety of cancers and induces the activities of matrix metalloproteinase (MMP)-2 and MMP-9. It has been reported that OPN induce matrix metalloproteinase (MMP)-2 and MMP-9 activations through nuclear factor kappaB (NF-κB)-mediated signaling pathways. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells, and provide clues about the possible functions of IκB kinase (IKK) in OPN-induced MMP-2/9 activitions by nuclear factor kappaB (NF-KB)-mediated signaling pathways. Methods: Four sorts of OPN short-hairpin RNA (shRNA) recombinant plasmids were constructed and transfected into PC-3 cells. The most highly functional shRNA recombinant plasmid was selected by RT-PCR for further studies, then after the stablely transfected cell line were established, the different concentrations of IKK inhibitors were used to inhibit the activities of IKK-1 and IKK-2. The mRNA and protein expression levers of OPN, MMP-2 and MMP-9 were detected by real-time PCR and Western blot. MMP-2 and MMP-9 protein levels in the medium of the cells cultured by OPN were detected by ELISA. The different cell cycles, cell proliferation and invasion abilities of PC-3 cells were detected by flow cytometry, MTT and Transwell chamber assays, respectively. The tumor formation assay were used to observe the growth situation of PC-3 cells in vivo. Results:The most highly functional shRNA recombinant plasmid (PGPU6/GFP/Neo-OPN2) was selected by RT-PCR assay. It can obviously inhibit OPN expression in PC-3 cells and the stablely transfected cell line, PCs, PC/Vect and PC/OPN2 were established. Compared with untreated cells(PCs), the protein expression levers of OPN, MMP-2 and MMP-9 in PC-3 cells transfected with recombinant plasmids (PC/OPN2) were reduced by 55.22%,51.71% and 28.35%, respectively, thereby resulting in suppression of the proliferation, migration and invasion of PC/OPN2 cells. Moreover, the inhibition of IKK-2 inhibited the expressions of MMP-2 and MMP-9, while the inhibition of IKK-1 had no influence on the expressions of MMP-2 and MMP-9. Conclusions:A short hairpin RNA expression vector-mediated OPN gene silencing can not only inhibit OPN expression in PC-3 cells but also decrease the expression levels of MMP-2 and MMP-9. OPN shRNA recombinant plasmid can inhibit the malignant physiological behaviors of PC-3 cells and these processes are associated with the activities of MMP-2 and MMP-9. Moreover, IKK-2 may play a crucial role in the OPN-induced activations of MMP-2 and MMP-9 via NF-κB mediated signaling pathways.
引文
1. J.T. Buijs, G. van der Pluijm, Osteotropic cancers:from primary tumor to bone, Cancer Lett.273 (2009) 177-193.
2. G.N. Thalmann, R.A. Sikes, R.E. Devoll, J.A. Kiefer, R. Markwalder, I. Klima, C.M. Farach-Carson, U.E. Studer, L.W. Chung, Osteopontin:possible role in prostate cancer progression, Clin. Cancer Res.5 (1999) 2271-2277.
3. H. Singhal, D.S. Bautista, K.S. Tonkin, F.P. O'Malley, A.B. Tuck, A.F. Chambers, J.F. Harris, Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival, Clin. Cancer 3 (1997) 605-611.
4. D. Agrawal, T. Chen, R. Irby, J. Quackenbush, A.F. Chambers, M. Szabo, A. Cantor, D. Coppola, T.J. Yeatman, Osteopontin identified as lead marker of colon cancer progression using pooled sample expression profiling, J. Natl. Cancer Inst. 94(2002)513-521.
5. P.S. Rudland, A. Platt-Higgins, M. El-Tanani, S. De Silva Rudland, R. Barraclough, J.H. Winstanley, R. Howitt, C.R. West, Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer, Cancer Res. 62(2002)3417-3427.
6. D. Coppola, M. Szabo, D. Boulware, P. Muraca, M. Alsarraj, A.F. Chambers, T.J. Yeatman, Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies, Clin. Cancer Res.10 (2004) 184-190.
7. S.R. Rittling, A.F. Chambers, Role of osteopontin in tumour progression, Br. J. Cancer 90 (2004) 1877-1881.
8. A. Macri, A. Versaci, G. Lupo, G. Trimarchi, C. Tomasello, S. Loddo, G. Sfuncia, R. Caminiti, D. Teti, C. Famulari, Role of osteopontin in breast cancer patients, Tumori.95 (2009) 48-52.
9. Y.C. Fong, S.C. Liu, C.Y. Huang, T.M. Li, S.F. Hsu, S.T. Kao, F.J. Tsai, W.C. Chen, C.Y. Chen, C.H. Tang, Osteopontin increases lung cancer cells migration via activation of the alphavbeta3 integrin/FAK/Akt and NF-kappaB-dependent pathway, Lung Cancer 64 (2009) 263-270.
10. J. Zhang, K. Takahashi, F. Takahashi, K. Shimizu, F. Ohshita, Y. Kameda, K. Maeda, K. Nishio, Y. Fukuchi, Differential osteopontin expression in lung cancer, Cancer Lett.171 (2001)215-222.
11. M. Gong, Z. Lu, G. Fang, J. Bi, X. Xue, A small interfering RNA targeting osteopontin as gastric cancer therapeutics, Cancer Lett.272 (2008) 148-159.
12. A. Jain, D.A. McKnight, L.W. Fisher, E.B. Humphreys, L.A. Mangold, A.W. Partin, N.S. Fedarko, Small integrin-binding proteins as serum markers for prostate cancer detection, Clin. Cancer Res.15 (2009) 5199-5207.
13. A.B. Tuck, D.M. Arsenault, F.P. O'Malley, C. Hota, M.C. Ling, S.M. Wilson, A.F. Chambers, Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells, Oncogene 18 (1999) 4237-4246.
14. H. Adwan, T.J. Bauerle, M.R. Berger, Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells, Cancer Gene Ther.11 (2004) 109-120.
15. H. Adwan, T. Bauerle, Y. Najajreh, V. Elazer, G. Golomb, M.R. Berger, Decreased levels of osteopontin and bone sialoprotein II are correlated with reduced proliferation, colony formation, and migration of GFP-MDA-MB-231 cells, Int. J. Oncol.24 (2004) 1235-1234.
16. T. Yamate, K. Kohri, T. Umekawa, M. Iguchi, T. Kurita, Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell, J. Urol.160 (1998) 1506-1512.
17. H. Nemoto, S.R. Rittling, H. Yoshitake, K. Furuya, T. Amagasa, K. Tsuji, A. Nifuji, D.T. Denhardt, M. Noda, Osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues, J. Bone Miner. Res.16 (2001) 652-659.
18. Y. Wu, D.T. Denhardt, S.R. Rittling, Osteopontin is required for full expression of the transformed phenotype by the ras oncogene, Br. J. Cancer 83 (2000) 156-163.
19. B. Desai, M.J. Rogers, M.A. Chellaiah, Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells, Mol. Cancer 6 (2007) 18.
20. G.F. Weber, S. Akshar, M.J. Glimcher, H. Cantor, Receptor-Ligand interaction between CD44 and osteopontin (Eta-1), Science 271 (1996) 509-519.
21. D. Panda, G.C. Kundu, B.I. Lee, A. Peri, D. Fohl, I. Chackalaparampil, B.B. Mukherjee, X.D. Li, D.C. Mukherjee, S. Seides, J. Rosenberg, K. Stark, A.B. Mukherjee, Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty, Proc. Natl. Acad. Sci. U.S. A.94(1997)9308-9313.
22. Z. Mi, H. Guo, M.B. Russell, Y. Liu, B.A. Sullenger, P.C. Kuo, RNA Aptamer Blockade of Osteopontin Inhibits Growth and Metastasis of MDA-MB231 Breast Cancer Cells, Mol Ther.17 (2009) 153-161.
23. B. Desai, T. Ma, J. Zhu, M.A. Chellaiah, Characterization of the expression of variant and standard CD44 in prostate cancer cells:identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface, J. Cell Biochem.108 (2009) 272-284.
24. G. Castellano, G. Malaponte, M.C. Mazzarino, M. Figini, F. Marchese, P. Gangemi, S. Travali, F. Stivala, S. Canevari, M. Libra. Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression, Clin. Cancer Res.14 (2008) 7470-7480.
25. S. Philip, A. Bulbule, G.C. Kundu, Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappaB-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells, J. Biol. Chem.276 (2001) 44926-44935.
26. H. Rangaswami, A. Bulbule, G.C. Kundu, Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/I kappaBalpha kinase-dependent nuclear factor kappa B-mediated promatrix metalloproteinase-9 activation, J. Biol. Chem.279 (2004) 38921-38935.
27. A.C. Khodavirdi, Z. Song, S. Yang, C. Zhong, S. Wang, H. Wu, C. Pritchard, P.S. Nelson, P. Roy-Burman, Increased expression of osteopontin contributes to the progression of prostate cancer, Cancer Res.66 (2006) 883-888.
28. F. Mercurio, H. Zhu, B.W. Murray, A. Shevchenko, B.L. Bennett, J. Li, D.B. Young, M. Barbosa, M. Mann, A. Manning, A. Rao, IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation, Science 278 (1997) 860-866.
29. C P. Paul, P D. Good, I. Winer, et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol,2002,20(5):505-508.
30. T R. Brummelkamp, R. Bernards, R. Agami. A system for stable expression of short interfering RNAs in mammalian cells. Science,2002,296(5567):550-553.
1. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer,2004,90(10):1877-1881.
2. Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res, 2002,62(12):3417-3427.
3. Shimada Y, Watanabe G, Kawamura J, et al. Clinical significance of osteopontin in esophageal squamous cell carcinoma:comparison with common tumor markers. Oncology,2005,68(2-3):285-292.
4. Desai B, Rogers MJ, Chellaiah MA. Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells. Mol Cancer,2007,6:18.
5. Jain A, McKnight DA, Fisher LW, et al. Small integrin-binding proteins as serum markers for prostate cancer detection. Clin Cancer Res,2009,15(16):5199-5207.
6. Castellano G, Malaponte G, Mazzarino MC, et al. Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin Cancer Res,2008,14(22):7470-7480.
7. Thalmann GN, Sikes RA, Devoll RE, et al. Osteopontin:possible role in prostate cancer progression. Clin Cancer Res,1999,5(8):2271-2277.
8. Philip S, Kundu G C. Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through I kappa B alpha/IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem,2003,278(16):14487-14497.
9. Rangaswami H, Bulbule A, Kundu G C. Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/I kappa B alpha kinase-dependent nuclear factor kappa B-mediated promatrix metalloproteinase-9 activation. J Biol Chem,2004,279(37)38921-38935.
10. Desai B, Ma T, Zhu J, et al. Characterization of the expression of variant and standard CD44 in prostate cancer cells:identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface. J Cell Biochem,2009,108(1):272-284.
11. Denhardt DT, Guo X. Osteopontin:a protein with diverse functions. FASEB J, 1993,7(15):1475-1482.
12. Prince, CW. Secondary structure predictions for rat osteopontin. Connect Tissue Res,1989,21(1-4):15-20.
13. Dai J, Peng L, Fan K, et al. Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene,2009,28(38): 3412-3422.
14.崔伯康,张昌卿,张颖,等.骨桥蛋白检测对预测肝细胞癌转移复发的意义.癌症,2006,25:876-978.
15. Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res,2004,10(1 Pt 1):184-190.
16. Gardner H A, Berse B, Senger D R. Specific reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat 1 fibroblasts. Oncogene,1994,9(8):2321-2326.
17. Paul C P, Good P D, Winer I, et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol,2002,20(5):505-508.
18. Brummelkamp T R, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science,2002,296(5567):550-553.
19. Wadhwa R, Kaul S C, Miyagishi M, et al. Know-how of RNA interference and its applications in research and therapy. Mutat Res,2004,567(1):71-84.
1. Rittling SR, Chambers AF. Role of osteopontin in tumor progression. Br J Cancer, 2004,90(10):1877-1881
2. Wai PY, Kuo PC. The role of osteopontin in tumor metastasis. J Surg Res,2004, 121 (2):228-241
3. Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res, 2002,62 (12):3417-3427
4. Shimada Y, Watanabe G, Kawamura J, et al. Clinical significance of osteopontin in esophageal squamous cell carcinoma:comparison with common tumor markers. Oncology,2005,68 (2-3):285-292
5. Coppola D, Szabo M, Boulware D et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res,2004,10:184-190.
6. Adwan H, Bauerle TJ, Berger MR. Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther,2004,11:109-120.
7. Mukhopadhyay R, Price JE. Stable expression of antisense osteopontin inhibits the growth of human breast cancer cells. Proc Am Assoc Cancer Res,90th Annual Meeting, Philadelphia, PA,1999,40:448.
8. Jain A, McKnight DA, Fisher LW, et al. Small integrin-binding proteins as serum markers for prostate cancer detection, Clin. Cancer Res.15 (2009) 5199-5207.
9. Butler WT. The nature and significance of osteopontin, Connect. Tissue Res.23 (1989) 123-136.
10. Hirama M, Takahashi F, Takahashi K, et al. Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth, Cancer Lett. 198(2003)107-117.
11. Denhardt DT, Guo X. Osteopontin:a protein with diverse functions, FASEB J.7 (1993) 1475-1482.
12. Naqvi AR, Islan MN, Choudhury NR, et al. The fascinating world of RNA interference, Int. J. Biol. Sci.5 (2009) 97-117.
13. Song J, Giang A, Lu Y, Pang S, Chiu R, Multiple shRNA expressing vector enhances efficiency of gene silencing, BMB Rep.41 (2008) 358-362.
14. Chang H. RNAi-mediated knockdown of target genes:a promising strategy for pancreatic cancer research, Cancer Gene Ther.14 (2007) 677-685.
15. Haasnoot J, Westerhout EM, Berkhout B. RNA interference against viruses:strike and counterstrike, Nat. Biotechnol.25 (2007) 1435-1443.
16. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells, Science 296 (2002) 550-553.
17. Dave RS, Pomerantz RJ. RNA interference:on the road to an alternate therapeutic strategy, Rev. Med. Virol.13 (2003) 373-385.
18. Tuck AB, Hota C, Wilson SM, Chambers AF. Osteopontin induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways, Oncogene 22 (2003) 1198-1205.
19. Tuck AB, Elliott BE, Hota C, et al. Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met), J. Cell Biochem.78 (2000) 465-475.
20. Lin YH, Huang CJ, Chao JR, et al. Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor, Mol. Cell Biol.20 (2000) 2734-2742.
21. Zhu B, Suzuki K, Goldberg HA, et al. Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin, J. Cell Physiol.198 (2004) 155-167.
22. Teramoto H, Castellone MD, Malek RL, et al. Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12, Oncogene 24 (2005) 489-501.
23. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells, J. Biol. Chem.278 (2003) 28593-28606.
24. Ogata T, Ueyama T, Nomura T, et al. Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway, Biochem. Biophys. Res. Commun. 359 (2007) 341-347.
25. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells, J. Biol.Chem.279 (2004) 11051-11064.
26. Desai B, Rogers MJ, Chellaiah MA. Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells, Mol Cancer,2007,6:18.
1. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer,2004,90(10):1877-1881.
2. Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res, 2002,62(12):3417-3427.
3. Shimada Y, Watanabe G, Kawamura J, et al. Clinical significance of osteopontin in esophageal squamous cell carcinoma:comparison with common tumor markers. Oncology,2005,68(2-3):285-292.
4. Desai B, Rogers MJ, Chellaiah MA. Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells. Mol Cancer,2007,6:18.
5. Jain A, McKnight DA, Fisher LW, et al. Small integrin-binding proteins as serum markers for prostate cancer detection. Clin Cancer Res,2009,15(16):5199-5207.
6. Castellano G, Malaponte G, Mazzarino MC, et al. Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin Cancer Res,2008,14(22):7470-7480.
7. Thalmann GN, Sikes RA, Devoll RE, et al. Osteopontin:possible role in prostate cancer progression. Clin Cancer Res,1999,5(8):2271-2277.
8. Philip S, Kundu G C. Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through I kappa B alpha/IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem,2003,278(16):14487-14497.
9. Rangaswami H, Bulbule A, Kundu G C. Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/I kappa B alpha kinase-dependent nuclear factor kappa B-mediated promatrix metalloproteinase-9 activation. J Biol Chem,2004,279(37)38921-38935.
10. Desai B, Ma T, Zhu J, et al. Characterization of the expression of variant and standard CD44 in prostate cancer cells:identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface. J Cell Biochem,2009,108(1):272-284.
11. Mercurio F, Zhu H, Murray B W, et al. IKK-1 and IKK-2:cytokine-activated I kappa B kinases essential for NF-kappa B activation. Science, 1997,278(5339):860-866.
12. Scorilas A, Karameris A, Arnogiannaki N, et al. Overexpression of matrix-metalloproteinase-9 in human breast cancer:a potential favourable indicator in node-negative patients. Br J Cancer,2001,84(11):1488-1496.
13. Eferl R, Wagner EF. AP-1:a double-edged sword in tumorigenesis. Nat Rev Cancer.2003 Nov;3(11):859-868
14. Denhardt DT, Mistretta D, Chambers AF. Transcriptional regulation of osteopontin and the metastatic phenotype:evidence for a Ras-activated enhancer in the human OPN promoter. Clin Exp Metastasis.2003;20(1):77-84
15. Chellaiah MA, Kizer N, Biswas R. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol Biol Cell.2003 Jan;14(1):173-189.
16. Ellen M. Osteopontin:a bridge between bone and the immune systerm. J Clin Invest.2003 Jul;112(2):147-149.
17. Takagi H, Suzuma K, Otani. A Role of vitronectin receptortype integrins and osteopontin in ischemia-induced retinal neovascularization. Jpn J Ophthalmol. 2002 May-Jun;46(3):270-278.
18. Pan HW, Ou YH, Peng SY. Overexpression of osteopontin is associated with intrahepatic metastasis,early recurrence,and poorer prognosis of surgically resected hepatocellular carcinoma. Cancer.2003 Jul 1;98(1):119-127.
19. Hotte SJ, Winquist EW, Stitt L. Plasma osteopontin:associations with survival and metastasis to bone in men with hormonerefractory prostate carcinoma. Cancer. 2002 Aug 1;95(3):506-512.
20. Takahashi F, Akutagawa S, Fukumoto H. Osteopontin induces angiogenesis of murine neuroblastoma cells in mice. Int J Cancer.2002 Apr 10;98(5):707-712.
21. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto SRel/NF-kappa B/I kappa B family:intimate tales of association and dissociationGenes Dev.1995 Nov 15;9(22):2723-2735
22. Baeuerle PA, Henkel T.Function and activation of NF-kappa B in the immune system.Annu Rev Immunol.1994; 12:141-179.
23. Baeuerle PA, Baltimore D.Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.Cell.1988 Apr 22;53(2):211-217.
24. Baeuerle PA, Baltimore D.A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev.1989 Nov,3(11):1689-1698.
25. Thompson JE, Phillips RJ, Erdjument-Bromage H, et al. I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell.1995 Feb 24;80(4):573-582.
26. Gilmore TD, Morin PJ.The I kappa B proteins:members of a multifunctional family. Trends Genet.1993 Dec;9(12):427-433.
27. Scherer DC, Brockman JA, Chen Z, et al. Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. Proc Natl Acad Sci U S A.1995 Nov 21;92(24):11259-11263.
28. Schouten GJ, Vertegaal AC, Whitesiide ST, Israel A, et al. IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase. EMBO J.1997 Jun 2;16(11):3133-3144.
29. Regnier CH, Song HY, Gao X, et al. Identification and characterization of an IkappaB kinase. Cell.1997 Jul 25;90(2):373-383.
1. Sodek J, Ganss B, Mc Kee MD. Osteopontin 2000(3).2
2. Sodek J, Zhu B, Huynh MH. Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC Connect Tissue Res.2002;43(2-3):308-319.
3. Senger D R, Wirth D F and Hynes R O.Transformed mammalian cells secrete specific proteins and phosphoproteins.Cell,1979,16(4):885-893
4. Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Acad Sci USA 1986; 83:8819-8823.
5. Weber GF. The metastasis gene osteopontin:a candidate target for cancer therapy. Biochim Biophys Acta.2001 Dec 28;1552(2):61-85.
6. Xie Y, Sakatsume M, Nishi S. Expression, roles, receptors, and regulation of osteopontin in the kidney Kidney Int.2001 Nov;60(5):1645-1657.
7. Ellen M. Osteopontin:a bridge between bone and the immune systerm J Clin Invest.2003 Jul;112(2):147-149.
8. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer. 2004 May 17;90(10):1877-1881.
9. You J, Reilly GC, Zhen X. Osteopontin gene regulation by osciallatory fluid flow via intracellular calcium mobilizaion and activation of mitogenactivated protein kinase in MC3T3-E1 osteoblasts. J Biol Chem.2001 Apr 20;276(16):13365-13371.
10. Chellaiah MA, Kizer N, Biswas R. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol Biol Cell.2003 Jan;14(1):173-189.
11.L IAW L, B IRK DE, BALLAS CB. Altered wound healing in mice lacking a functional osteopontin gene (Spp1).J Clin Invest.1998 Apr 1;101(7):1468-1478.
12. Meerwitch K, Bergeron F,Leblond L. A novel RGD antagonist that targets bothαvβ3 and α5β1 induces apoptosis of angiogenic endothelial cells on type Ⅰ collagen Vascul Pharmacol.2003 Feb;40(2):77-89.
13. Yee D, McGuire SE, Brunner N, et al. Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. Hum Gene Ther 1996;7:1251-1257.
14. Lin YH, Yang-Yen HF. The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway.J Biol Chem.2001 Dec 7;276(49):46024-46030.
15. Chellaiah MA, Kizer N, Biswas R. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol Biol Cell.2003 Jan;14(1):173-189.
16. Desai B, Rogers MJ, Chellaiah MA. Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells. Mol Cancer 6 (2007) 18.
17. Weber GF, Akshar S, Glimcher MJ, et al. Receptor-Ligand interaction between CD44 and osteopontin (Eta-1). Science 271 (1996) 509-519.
18. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappaB-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J. Biol. Chem.276 (2001) 44926-44935.
19. Egblad M, Werb Z. New functions for the matrix metalloprteinas in cancer progression. Nat Rev Cancer.2002 Mar;2(3):161-174.
20. Denhardt DT, Mistretta D, Chambers AF. Transcriptional regulation of osteopontin and the metastatic phenotype:evidence for a Ras-activated enhancer in the human OPN promoter. Clin Exp Metastasis.2003;20(1):77-84.
21. Prols F, Loser B, Marx M. Differential expression of osteopontin, Pc4 and CEEC5, a novel mRNA species, during in vitro angiongenesis. Exp Cell Res.1998 Feb 25;239(1):1-10.
22. Wai PY,Kuo PC. The role of osteopontin in tumor metastasis J Surg Res.2004 Oct;121(2):228-241.
23. Eferl R, Wagner EF. AP-1:a double-edged sword in tumorigenesis. Nat Rev Cancer. 2003 Nov;3(11):859-868.
24. Ding Q, Stewart J, Charles JW. Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain:differences in integrin signaling during cell adhesion to osteopontin versus vitronectin Cancer Res.2002 Sep 15;62(18):5336-5343.
25. Zhang J, Talahashi K, Talahashi F. Differential osteopontin expression in lung cancer.Cancer Lett.2001 Oct 10;171(2):215-222.
26. Furger KA, Allan AL, Wilson SM. Betaβ3 integrin expression increases breast carcinoma cell responsiveness to the malignancy-enhancing effects of osteopontin. Mol Cancer Res.2003 Sep;1(11):810-819.
27. Hotte SJ, Winquist EW, Stitt L. Plasma osteopontin:associations with survival and metastasis to bone in men with hormonerefractory prostate carcinoma. Cancer. 2002 Aug 1;95(3):506-512.
28. Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin. Cancer Res.10(2004) 184-190.
29. Yamate T, Kohri K, Umekawa T, et al. Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell. J. Urol.160(1998)1506-1512.
30.贺斌,刘银坤,汤钊猷.肝癌中骨桥蛋白表达的临床意义.中华肝脏病杂志.1998(4).
31.Singhal H, Bautista DS, Tonkin KS, et al.Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin. Cancer 3 (1997) 605-611.
32. Strom A, Franzen A, Wangnerud C, et al. Altered vascular remodeling in osteopontin-deficient atherosclerotic mice. J Vasc Res.2004,41(4):314-22.
33. Guo-Xin Zhang, Zhi-Quan Zhao, Hong-Di Wang, Bo Hao. Enhancement of osteopontin expression in HepG2 cells by epidermal growth factor via phosphatidylinositol 3-kinase signaling pathway. World J Gastroenterol.2004 Jan 15;10(2):205-208.
34. Panda D, Kundu GC, Lee BI, et al. Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty. Proc. Natl. Acad. Sci. U. S. A.94 (1997) 9308-9313.
35. Khodavirdi AC, Song Z, Yang S, et al. Increased expression of osteopontin contributes to the progression of prostate cancer. Cancer Res.66 (2006) 883-888.
36. Zhang R, Xu Y, Ekman N. Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway. J Biol Chem.2003 Dec 19;278(51): 51267-51276.
37. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappaB-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J. Biol. Chem.276 (2001) 44926-44935.
38. Rangaswami H, Bulbule A, Kundu GC. Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/I kappaBalpha kinase-dependent nuclear factor kappa B-mediated promatrix metalloproteinase-9 activation. J. Biol. Chem.279 (2004) 38921-38935.
39. Ahmad SA, Jung YD, Liu W. The role of the microenvironment and intercellular cross-talk in tumor angiogenesis. Semin Cancer Biol.2002 Apr; 12(2):105-112.
40. Takagi H, Suzuma K, Otani A. Role of vitronectin receptortype integrins and osteopontin in ischemia-induced retinal neovascularization.Jpn J Ophthalmol. 2002 May-Jun;46(3):270-278.
41. Romer J, Nielsen BS, Ploug M. The urokinase receptor as a potential target in cancer therapy. Curr Pharm Des.2004;10(19):2359-2376.
42. Thalmann GN, Sikes RA, Devoll RE, et al. Osteopontin:possible role in prostate cancer progression. Clin Cancer Res.5 (1999) 2271-2277.
43. Castellone G, Malaponte G, Mazzarino MC, et al. Activation of the osteopontin/ matrix metalloproteinase-9 pathway correlates with prostate cancer progression. Clin. Cancer Res.14 (2008) 7470-7480.
44. Tuck AB, Arsenault DM, O'Malley FP, et al. Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 18 (1999) 4237-4246.
45. Adwa n H, Bauerle TJ, Berger MR, Downregulation of osteopontin and bone sialoprotein Ⅱ is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther.11 (2004) 109-120.
46. Adwan H, Bauerle T, Najajreh Y, et al.Decreased levels of osteopontin and bone sialoprotein Ⅱ are correlated with reduced proliferation, colony formation, and migration of GFP-MDA-MB-231 cells. Int. J. Oncol.24 (2004) 1235-1234.
47. Nemoto H, Rittling SR, Yoshitake H, et al. Osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues. J. Bone Miner. Res. 16(2001)652-659.
48. Wu Y, Denhardt DT, Rittling SR. Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br. J. Cancer 83 (2000) 156-163.
49. Allan AL, George R, Vantyghem SA, et al. Role of the integrin-binding protein osteopontin in lymphatic metastasis of breast cancer. Am J Pathol.2006 Jul;169(1):233-246.
50. Yu Q, Stamenkovic I. Cell surface-loccalizaed matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev.2000 Jan 15; 14(2):163-176.
51. Deryugina El, Ratnikov B, Monosov E. MT1-MMP initiates activation of pro-MMP-2 and integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells. Exp Cell Res.2001 Feb 15;263(2):209-223.
52. Mi Z, Guo H, Russell MB, et al. RNA Aptamer Blockade of Osteopontin Inhibits Growth and Metastasis of MDA-MB231 Breast Cancer Cells. Mol Ther.17 (2009) 153-161.