Background

[PubMed]

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3).

Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (4-7). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (8). There are four members of endogenous tissue inhibitors of metalloproteinases (TIMPs), which regulate the activity of MMPs leading to inhibition of tumor growth and metastasis (9, 10). TIMP-2 (TIMP2) is a bifunctional inhibitor of angiogenesis by inhibition of proteinase activity of MMPs and endothelial cell proliferation via binding to α₃β₁ (the N-terminal domain) and by MMP-independent anti-angiogenic activity (the C-terminal domain) (11, 12). Kang et al. (13) fused the N-terminal domain of TIMP2 to the C-terminus of human serum albumin (HSA) to form HSA/TIMP2 fusion protein (HSA-TIMP2), which is readily secreted by the transfected yeast Saccharomyces cerevisiae. HSA-TIMP2 retains its anti-angiogenic activity at the C-terminal domain with little MMP inhibitory activity at the N-terminal domain. Lee et al. (14) have evaluated Cy5.5-HSA/TIMP2 (Cy5.5-HSA-TIMP2) for in vivo near-infrared (NIR) fluorescence imaging of rat prostate MLL tumors in nude mice showing maximum tumor accumulation at 2 d after injection.

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (15). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α_vβ₃ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (16-21). Expression of the α_vβ₃ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α_vβ₃. The α_vβ₃ antagonists are being studied as anti-tumor and anti-angiogenic agents (18, 22, 23). Various radiolabeled RGD peptides (antagonists) have been introduced for imaging of tumors and tumor angiogenesis (24). Choi et al. (25) conjugated multiple c(RGDfK) peptides to HSA-TIMP2 to enhance the binding capacity of the protein (RGD-HSA-TIMP2) to tumors and their vasculatures. ¹²³I-RGD-HSA-TIMP2 and ¹²³I-HSA-TIMP2 have been studied as potential single-photon emission computed tomography (SPECT) probes for imaging α_vβ₃ receptors in nude mice bearing human glioblastoma U87MG tumors.

Synthesis

[PubMed]

HSA-TIMP2 (0.23 µmol) and c[RGDfK(COCH₂SH)] (1.39 µmol) were incubated with N-succinimidyl iodoacetate (3.52 µmol) to form a thioether linkage between the RGD peptide and HSA-TIMP2 protein (25). RGD-HSA-TIMP2 was purified with column chromatography. The molecular weights of RGD-HSA-TIMP2 and HSA-TIMP2 were calculated to be 88.42 kDa and 92.78 kDa with mass spectroscopy, respectively. There were 6 RGD molecules per RGD-HSA-TIMP2. ¹²³I-RGD-HSA-TIMP2 and ¹²³I-HSA-TIMP2 were prepared with the Iodogen method by incubation of ~11 nmol protein with 185 MBq (5 mCi) [¹²³I]NaI for 20 min at room temperature. The radiochemical yields were 45%–50%. The specific activities of ¹²³I-RGD-HSA-TIMP2 and ¹²³I-HSA-TIMP2 were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Flow cytometry analysis showed that 45% and 91% of U89MG cells were positive after incubation with HSA-TIMP2-FITC and RGD-HSA-TIMP2-FITC for 24 h at 4°C (25), respectively. Confocal microscopy showed internalization of fluorescence activity inside the cells incubated with 1.6 nM RGD-HSA-TIMP2-FITC for 3 h at 37°C but not with HSA-TIMP2-FITC. No blocking studies were performed with unlabeled RGD-HSA-TIMP2.

In vitro cell viability of U87MG was assessed after incubation with various concentrations (0.11–3.2 nM) of RGD, HSA-TIMP2, and RGD-HSA-TIMP2 for 24 h at 37°C (25). The cell viability values with 3.2 nM RGD, HSA-TIMP2, and RGD-HSA-TIMP2 were 92.5 ± 4.0%, 73.5 ± 7.9% (P < 0.05 versus RGD), and 66.7 ± 0.6% (P < 0.001 versus RGD), respectively.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

1.

Bosman F.T., Stamenkovic I. Functional structure and composition of the extracellular matrix. J Pathol. 2003;200(4):423–8. [PubMed: 12845610]

2.

Jiang W.G., Puntis M.C., Hallett M.B. Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br J Surg. 1994;81(11):1576–90. [PubMed: 7827878]

3.

Albelda S.M. Role of integrins and other cell adhesion molecules in tumor progression and metastasis. Lab Invest. 1993;68(1):4–17. [PubMed: 8423675]

4.

Keppler D., Sameni M., Moin K., Mikkelsen T., Diglio C.A., Sloane B.F. Tumor progression and angiogenesis: cathepsin B & Co. Biochem Cell Biol. 1996;74(6):799–810. [PubMed: 9164649]

5.

Liu J., Sukhova G.K., Sun J.S., Xu W.H., Libby P., Shi G.P. Lysosomal cysteine proteases in atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24(8):1359–66. [PubMed: 15178558]

6.

Berchem G., Glondu M., Gleizes M., Brouillet J.P., Vignon F., Garcia M., Liaudet-Coopman E. Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis. Oncogene. 2002;21(38):5951–5. [PubMed: 12185597]

7.

Brix, K., A. Dunkhorst, K. Mayer, and S. Jordans, Cysteine cathepsins: Cellular roadmap to different functions. Biochimie, 2007. [PubMed: 17825974]

8.

Deryugina E.I., Quigley J.P. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 2006;25(1):9–34. [PubMed: 16680569]

9.

Baker A.H., Edwards D.R., Murphy G. Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cell Sci. 2002;115(Pt 19):3719–27. [PubMed: 12235282]

10.

Jiang Y., Goldberg I.D., Shi Y.E. Complex roles of tissue inhibitors of metalloproteinases in cancer. Oncogene. 2002;21(14):2245–52. [PubMed: 11948407]

11.

Fernandez C.A., Butterfield C., Jackson G., Moses M.A. Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor. J Biol Chem. 2003;278(42):40989–95. [PubMed: 12900406]

12.

Seo D.W., Li H., Guedez L., Wingfield P.T., Diaz T., Salloum R., Wei B.Y., Stetler-Stevenson W.G. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell. 2003;114(2):171–80. [PubMed: 12887919]

13.

Kang W.K., Park E.K., Lee H.S., Park B.Y., Chang J.Y., Kim M.Y., Kang H.A., Kim J.Y. A biologically active angiogenesis inhibitor, human serum albumin-TIMP-2 fusion protein, secreted from Saccharomyces cerevisiae. Protein Expr Purif. 2007;53(2):331–8. [PubMed: 17368046]

14.

Lee M.S., Kim Y.H., Kim Y.J., Kwon S.H., Bang J.K., Lee S.M., Song Y.S., Hahm D.H., Shim I., Han D., Her S. Pharmacokinetics and biodistribution of human serum albumin-TIMP-2 fusion protein using near-infrared optical imaging. J Pharm Pharm Sci. 2011;14(3):368–77. [PubMed: 21962154]

15.

Hynes R.O. A reevaluation of integrins as regulators of angiogenesis. Nat Med. 2002;8(9):918–21. [PubMed: 12205444]

16.

Grzesik W.J. Integrins and bone--cell adhesion and beyond. Arch Immunol Ther Exp (Warsz). 1997;45(4):271–5. [PubMed: 9523000]

17.

Jin H., Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]

18.

Kumar C.C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4(2):123–31. [PubMed: 12558065]

19.

Ruegg C., Dormond O., Foletti A. Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target? Endothelium. 2002;9(3):151–60. [PubMed: 12380640]

20.

Varner J.A., Cheresh D.A. Integrins and cancer. Curr Opin Cell Biol. 1996;8(5):724–30. [PubMed: 8939661]

21.

Wilder R.L. Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann Rheum Dis. 2002;61 Suppl 2:ii96–9. [PMC free article: PMC1766704] [PubMed: 12379637]

22.

Kerr J.S., Mousa S.A., Slee A.M. Alpha(v)beta(3) integrin in angiogenesis and restenosis. Drug News Perspect. 2001;14(3):143–50. [PubMed: 12819820]

23.

Mousa S.A. alphav Vitronectin receptors in vascular-mediated disorders. Med Res Rev. 2003;23(2):190–9. [PubMed: 12500288]

24.

Haubner R., Beer A.J., Wang H., Chen X. Positron emission tomography tracers for imaging angiogenesis. Eur J Nucl Med Mol Imaging. 2010;37 Suppl 1:S86–103. [PMC free article: PMC3629959] [PubMed: 20559632]

25.

Choi N., Kim S.M., Hong K.S., Cho G., Cho J.H., Lee C., Ryu E.K. The use of the fusion protein RGD-HSA-TIMP2 as a tumor targeting imaging probe for SPECT and PET. Biomaterials. 2011;32(29):7151–8. [PubMed: 21719102]