Inhibition of colon cancer growth and metastasis by NK4 gene repetitive delivery in mice

https://doi.org/10.1016/j.bbrc.2007.04.098Get rights and content

Abstract

NK4, originally prepared as a competitive antagonist for hepatocyte growth factor (HGF), is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. When the expression plasmid for NK4 gene was administered into mice by hydrodynamics-based delivery, the repetitive increase in the plasma NK4 protein level was achieved by repetitive administration of NK4 gene. Mice were subcutaneously implanted with colon cancer cells and weekly given with the NK4 plasmid. The repetitive delivery and expression of NK4 gene inhibited angiogenesis and invasiveness of colon cancer cells in subcutaneous tumor tissue and this was associated with suppression of primary tumor growth. By fifty days after tumor implantation, cancer cells naturally metastasized to the liver, whereas NK4 gene expression potently inhibited liver metastasis. Inhibition of the HGF-Met receptor pathway and tumor angiogenesis by NK4 gene expression has potential therapeutic value toward inhibition of invasion, growth, and metastasis of colon cancer.

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Materials and methods

Recombinant materials and measurement of NK4. Recombinant human HGF and NK4 were respectively purified from culture media of CHO cells, which stably secrete human HGF and NK4 [6], [18]. Concentration of NK4 in the plasma was determined using enzyme-linked immunosorbent assay (ELISA) kit for human HGF (B-Bridge International Inc., CA, USA). Because this ELISA system does not cross-react with mouse HGF, human NK4 can be specifically detectable in mouse tissues and plasma. Recombinant human NK4

Inhibition of HGF-induced cell scattering by NK4 expressed in COS-7 cells

We first tested whether NK4 expressed in mammalian cells using pCAGGS-NK4 plasmid would inhibit biological response driven by HGF-Met association. COS-7 cells were transfected with pCAGGS-NK4 plasmid and biological activity of NK4 in the conditioned medium was examined in cell scattering assay using MDCK cells (Fig. 1A). When 110 pM HGF was added to monolayer culture of MDCK cells, HGF induced dissociation and scattering of the cells (Fig. 1A(a) and (b)), however, recombinant NK4 inhibited MDCK

Discussion

Since HGF activates intracellular and extracellular events that lead to dissociation and invasion of cancer cells, the HGF-Met system is closely involved in acquisition of invasive and metastatic potentials [7], [8], [9], [10]. HGF decreases cadherin-mediated adhesiveness of cells [20], [21], and stimulates proteolytic breakdown of the extracellular matrix, through enhancing matrix metalloproteinase and urokinase-type plasminogen activator-dependent proteolytic network [7], [8], [9], [10], [11]

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Technology, Sports and Culture of Japan, and Grant for Cancer Research from the Ministry of Health, Labor, and Welfare of Japan.

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