Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells

Abstract

Urokinase-type plasminogen activator (u-PA) plays a key role in malignant tumor behavior. We have previously shown that the expression of high levels of u-PA mRNA in human hepatocellular carcinoma (HCC) biopsies was inversely correlated with the survival of the patients. In order to evaluate the involvement of u-PA in the invasive and infiltrating properties of HCC cells, the SKHep1C3 cell line was stably transfected with an expression vector containing the 5′ portion (257 bp) of u-PA cDNA in the antisense orientation. u-PA mRNA expression and its protein level and enzymatic activity were specifically inhibited in the antisense transfectants. A comparable inhibition of the u-PA receptor (u-PAR) mRNA and protein was also evidenced in the antisense transfected cells compared with the control ones. At the functional level, the SKHep1C3-AS cells showed a significant reduction in proliferation, Matrigel invasion, and motility assays compared to parental and vector-alone cells. These results indicate that u-PA is an essential factor in the growth and invasiveness of human hepatocarcinoma cells. Antisense u-PA strategy might be a potential approach to reduce tumor growth as well as the invasive capacity of the malignant cells in HCC.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Chen PJ, Chen DS, Lai MY, et al. Clonal origin of recurrent hepatocellular carcinomas. Gastroenterology. 1989;96:527–529.

    Article  CAS  Google Scholar 

  2. Yamamoto T, Kajino K, Kudo M, Sasaki Y, Arakawa Y, Hino O . Determination of the clonal origin of multiple human hepatocellular carcinomas by cloning and polymerase chain reaction of the integrated hepatitis B virus DNA. Hepatology. 1999;29:1446–1452.

    Article  CAS  Google Scholar 

  3. Mignatti P, Rifkin DB . Biology and biochemistry of proteinases in tumor invasion. Phys Rev. 1993;73(1):61–185.

    Google Scholar 

  4. Dano K, Romer J, Nielsen BS, et al. Cancer invasion and tissue remodeling — cooperation of protease systems and cell types. APMIS. 1999;107:120–127.

    Article  CAS  Google Scholar 

  5. Andreasan PA, Kioller L, Christensen L, Duffy MJ . The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 1997;72:1–22.

    Article  Google Scholar 

  6. Barlati S . Plasminogen activators and fibronectin fragments in tumor growth and signal transduction. Bull Inst Pasteur. 1994;92:269–275.

    CAS  Google Scholar 

  7. Schmitt M, Wilhelm OG, Reuning U, et al. The urokinase plasminogen activator system as a novel target for tumor therapy. Fibrinolysis Proteolysis. 2000;14:114–132.

    Article  CAS  Google Scholar 

  8. Blasi F . u-PA, u-PAR, PAI-1: key intersection of proteolytic, adhesive and chemotactic highways? Immunol Today. 1997;18:415–417.

    Article  CAS  Google Scholar 

  9. Chapman HA . Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr Opin Cell Biol. 1997;9:714–724.

    Article  CAS  Google Scholar 

  10. Dumler I, Stepanova V, Jerke U, et al. Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin. Curr Biol. 1999;9:1468–1476.

    Article  CAS  Google Scholar 

  11. De Petro G, Copeta A, Barlati S . Urokinase-type and tissue-type plasminogen activators as growth factors of human fibroblasts. Exp Cell Res. 1994;213:286–294.

    Article  CAS  Google Scholar 

  12. Kirchheimer JC, Wojta J, Christ G, Binder BR . Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line. Proc Natl Acad Sci USA. 1989;86:5424–5428.

    Article  CAS  Google Scholar 

  13. Fischer K, Lutz V, Wilhelm O, et al. Urokinase induces proliferation of human cancer cells: characterization of structural elements required for growth factor function. FEBS Lett. 1998;438:101–105.

    Article  CAS  Google Scholar 

  14. Schmitt M, Harbeck N, Thomssen C, et al. Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy. Thromb Haemost. 1997;78:285–296.

    Article  CAS  Google Scholar 

  15. De Petro G, Tavian D, Copeta A, Portolani N, Giulini SM, Barlati S . Expression of urokinase-type plasminogen activator (u-PA), u-PA receptor, and tissue-type PA messenger RNAs in human hepatocellular carcinoma. Cancer Res. 1998;58:2234–2239.

    CAS  PubMed  Google Scholar 

  16. Tavian D, De Petro G, Benetti A, Portolani N, Giulini SM, Barlati S . u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma. Int J Cancer. 2000;87:644–649.

    Article  CAS  Google Scholar 

  17. Barlati S, Zoppi N, Copeta A, Tavian D, De Petro G, Colombi M . Quantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections. Histol Histopathol. 1999;14:1231–1240.

    CAS  PubMed  Google Scholar 

  18. Tavian D, De Petro G, Colombi M, et al. RT-PCR detection of fibronectin EDA+ and EDB mRNA isoforms: molecular markers for hepatocellular carcinoma. Int J Cancer. 1994;56:820–825.

    Article  CAS  Google Scholar 

  19. Copeta A, Tavian D, Marchina E, De Petro G, Barlati S . Gene response of human skin fibroblasts to urokinase- and tissue-type plasminogen activators. Growth Factors. 2000;17:249–268.

    Article  CAS  Google Scholar 

  20. Raff T, van der Giet M, Endemann D, Wiederholt T, Paul M . Design and testing of β-actin primers for RT-PCR that do not co-amplify processed pseudogenes. BioTechniques. 1997;23:456–460.

    Article  CAS  Google Scholar 

  21. Bassi MT, Gasol E, Manzoni M, et al. Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc. Eur J Physiol. 2001;442:286–296.

    Article  CAS  Google Scholar 

  22. De Petro G, Tavian D, Marchina E, Barlati S . Induction of fibronectin mRNA by urokinase- and tissue-type plasminogen activator in human skin fibroblasts: different role of u-PA and t-PA at the fibronectin protein level. Biol Chem. 2002;383:177–187.

    Article  CAS  Google Scholar 

  23. Marchina E, De Petro G, Barlati S . Interaction of tissue-type plasminogen activators with fibronectin fragments. Fibrinolysis. 1993;7:51–57.

    Article  CAS  Google Scholar 

  24. Degrise B, Sier CF, Resnati M, Conese M, Blasi F . PAI-1 inhibits urokinase-induced chemotaxis by internalizing the urokinase receptor. FEBS Lett. 2001;505:49–254.

    Google Scholar 

  25. Resnati M, Pallavicini I, Wang JM, et al. The fibrinolytic receptor for urokinase activates the G protein–coupled chemotactic receptor FPRL1/LXA4R. Proc Natl Acad Sci USA. 2002;99:1359–1364.

    Article  CAS  Google Scholar 

  26. Haeckel C, Krueger S, Roessner A . Antisense inhibitor of urokinase: effect on malignancy in a human osteosarcoma cell line. Int J Cancer. 1998;77:153–160.

    Article  CAS  Google Scholar 

  27. Mohanam S, Jasti SL, Kondraganti SR, et al. Stable transfection of urokinase-type plasminogen activator antisense construct modulates invasion of human glioblastoma cells. Clin Cancer Res. 2001;7:2519–2526.

    CAS  PubMed  Google Scholar 

  28. Shetty S, Idell S . Urokinase induces expression of its own receptor in Beas2B lung epithelial cells. J Biol Chem. 2001;276:24549–24556.

    Article  CAS  Google Scholar 

  29. Montuori N, Mattiello A, Mancini A, et al. Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor through a post-transcriptional mechanism. FEBS Lett. 2001;508:379–384.

    Article  CAS  Google Scholar 

  30. Kook YH, Adamski J, Zelent A, Ossowski L . The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma on malignancy. EMBO J. 1994;13:3983–3991.

    Article  CAS  Google Scholar 

  31. Mohanam S, Chintala SK, GO Y, et al. In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor. Oncogene. 1997;14:1351–1359.

    Article  CAS  Google Scholar 

  32. Yu W, Kim J, Ossowski L . Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy. J Cell Biol. 1997;137:767–777.

    Article  CAS  Google Scholar 

  33. Waltz DA, Chapman HA . Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy. J Biol Chem. 1994;269:14746–14750.

    CAS  PubMed  Google Scholar 

  34. Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science. 1996;273:1551–1555.

    Article  CAS  Google Scholar 

  35. Aguirre-Ghiso JA, Liu D, Mignatti A, Kovalski K, Ossowski L . Urokinase receptor and fibronectin regulated the ERKMAPK to p38MAPK activity ratios that determine carcinoma cell proliferation or dormancy in vivo. Mol Biol Cell. 2001;12:863–879.

    Article  CAS  Google Scholar 

  36. Wei Y, Eble JA, Wang Z, Kreidberg JA, Chapman HA . Urokinase receptors promote β1 integrin function through interactions with integrin α3β1. Mol Biol Cell. 2001;12:2975–2986.

    Article  CAS  Google Scholar 

  37. Ossowski L . Plasminogen activator dependent pathways in the dissemination of human tumor cells in the chick embryo. Cell. 1988;52:321–328.

    Article  CAS  Google Scholar 

  38. Shetty S, Kumar A, Johnson A, Pueblitz S, Idell S . Urokinase receptor in human malignant mesothelioma cells: role in tumor cell mitogenesis and proteolysis. Am J Physiol. 1995;268:972–982.

    Google Scholar 

  39. Ma Z, Webb DJ, JO M, Gonias SL . Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells. J Cell Sci. 2001;114:3387–3396.

    CAS  PubMed  Google Scholar 

  40. Nakamura T . Structure and function of hepatocyte growth factor. Prog Growth Factor Res. 1991;3:67–85.

    Article  CAS  Google Scholar 

  41. Jeffers M, Rong S, Vande Woude GF . Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-Met signalling in human cells concomitant with induction of the urokinase proteolysis network. Mol Cell Biol. 1996;16:1115–1125.

    Article  CAS  Google Scholar 

  42. Jiang Y, Xu W, Lu J, He F, Yang X . Invasiveness of hepatocellular carcinoma cell lines: contribution of hepatocyte growth factor, c-met, and transcription factor Ets-1. Biochem Biophys Res Commun. 2001;286:1123–1130.

    Article  CAS  Google Scholar 

  43. Itoh T, Hayashi Y, Kanamaru T, et al. Clinical significance of urokinase-type plasminogen activator activity in hepatocellular carcinoma. J Gastroenterol Hepatol. 2000;15:422–430.

    Article  CAS  Google Scholar 

  44. Zheng Q, Tang ZY, Xue Q, Shi DR, Song HY, Tang HB . Invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor. J Cancer Res Clin Oncol. 2000;126:641–646.

    Article  CAS  Google Scholar 

  45. Zhou L, Hayashi Y, Itoh T, Wang W, Rui J, Itoh H . Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 and -2 in hepatocellular carcinoma. Pathol Int. 2000;50:392–397.

    Article  CAS  Google Scholar 

  46. Dubuisson L, Monvoisin A, Nielsen BS, Le Bail B, Bioulac-Sage P, Rosembaum J . Expression and cellular localization of the urokinase-type plasminogen activator and its receptor in human hepatocellular carcinoma. J Pathol. 2000;190:190–195.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Sergio Ferraboli for performing DNA sequencing, and Bruna Arici and Anna Alghisi for their invaluable technical assistance. Our thanks also go to Shelley Wintle (University of Brescia, Italy) for revising the English. This work was supported by Grants awarded by the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Centro Eccellenza IDET, Fondazione Cariplo and Consiglio Nazionale delle Ricerche, and MIUR in the context of Progetto Strategico Oncologia.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sergio Barlati.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tavian, D., Salvi, A., De Petro, G. et al. Stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells. Cancer Gene Ther 10, 112–120 (2003). https://doi.org/10.1038/sj.cgt.7700533

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700533

Keywords

Search

Quick links