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NK4 Gene Therapy Inhibits HGF/Met-Induced Growth of Human Cholangiocarcinoma Cells

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Abstract

Background and Objective

NK4, a competitive antagonist for hepatocyte growth factor (HGF) and the Met receptor, is a bifunctional molecule that acts as an HGF antagonist and an angiogenesis inhibitor. The objective of this study was to investigate the anti-tumor effects of NK4 on the cholangiocarcinoma (CCA) cell line HuCC-T1.

Methods

We assessed the effects of NK4 on proliferation, invasion, migration, and cell cycle progression in mock-transfected HuCC-T1 clones, empty-vector-transfected clones of HuCC-T1 (Hu-Em), and NK4-transfected clones of HuCC-T1 (Hu-NK4), with HuCC-T1 cells serving as the control cells. Correlated with these effects on cellular functions, the mRNA levels of cyclin D1 and cyclin A were monitored using reverse transcription (RT)-PCR and quantitative PCR, and the corresponding protein levels were monitored using Western blotting. In addition, Met phosphorylation and the activity of its important downstream signaling targets protein kinase B (Akt) and glycogen synthase kinase (GSK)-3β were evaluated by Western blotting.

Results

Our data indicate that cell proliferation, invasion, and cell cycle progression of the three types of clones were essentially the same, while these processes were stimulated by HGF in HuCC-T1 and Hu-Em cells, but not in Hu-NK4 cells. Moreover, when stimulated with HGF, the increases in mRNA levels of cyclin D1 and cyclin A were accompanied by corresponding increases in protein levels, and the phosphorylation of Met, Akt, and GSK-3β was upregulated in HuCC-T1 and Hu-Em cells, compared to the levels in the Hu-NK4 cells.

Conclusions

These findings suggest that NK4 gene therapy inhibits HGF/Met-induced growth of human CCA cells by arresting cell cycle progression. It also interferes with Met activation and the downstream phosphatidylinositol-3-kinase/Akt/GSK-3β signaling pathway.

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Acknowledgments

We are grateful to Prof. Toshikazu Nakamura and Prof. Kiyomasa Oka (Osaka University Medical School, Japan) for their generosity in providing the human NK4 expression plasmid pcDNA3/NK4. We also acknowledge the excellent technical assistance of Dr. Yanggang Yuan and Dr. Chenbo Ji (Institute of Pediatrics, Nanjing Medical University, China). This study was supported by grants from the Jiangsu Health Department (H200835) and the Natural Science Foundation of Jiangsu Province (BK2012871).

Conflict of interest

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Authors and Affiliations

  1. Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, 121 Jiang Jia Yuan, Nanjing, 210011, Jiangsu Province, China

    Xianxiu Ge, Yun Wang, Quanpeng Li, Hong Yu & Lin Miao

  2. The Clinical Laboratory of Nanjing Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu Province, China

    Youli Wang

Authors
  1. Xianxiu Ge
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  2. Youli Wang
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  3. Yun Wang
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  4. Quanpeng Li
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  5. Hong Yu
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  6. Lin Miao
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Corresponding author

Correspondence to Lin Miao.

Additional information

Xianxiu Ge and Youli Wang contributed equally to this work.

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Ge, X., Wang, Y., Wang, Y. et al. NK4 Gene Therapy Inhibits HGF/Met-Induced Growth of Human Cholangiocarcinoma Cells. Dig Dis Sci 58, 1636–1643 (2013). https://doi.org/10.1007/s10620-012-2523-7

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  • DOI: https://doi.org/10.1007/s10620-012-2523-7

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