Molecular Medicine

, Volume 14, Issue 7–8, pp 365–373 | Cite as

1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP)-Formulated, Immune-Stimulatory Vascular Endothelial Growth Factor A Small Interfering RNA (siRNA) Increases Antitumoral Efficacy in Murine Orthotopic Hepatocellular Carcinoma with Liver Fibrosis

  • Miroslaw Kornek
  • Veronika Lukacs-Kornek
  • Andreas Limmer
  • Esther Raskopf
  • Ursula Becker
  • Maren Klöckner
  • Tilman Sauerbruch
  • Volker Schmitz
Research Article


Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 µg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased β-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC.



This work was partly supported by a Deutsche Krebshilfe and a DFG grant to V.S. No conflicts of interest exist.

We thank Prof. P.A. Knolle, head of the Institute of Molecular Medicine and Experimental Immunology (IMMEI, Bonn, Germany) for continuous support of the project. Additionally we thank our practicum-student Bettina Schroll for her assistance during the tumor implantation surgery.


  1. 1.
    Anthony PP et al. (1978) The morphology of cirrhosis: recommendations on definition, nomenclature, and classification by a working group sponsored by the World Health Organization. J. Clin. Pathol. 31:395–414.CrossRefPubMedGoogle Scholar
  2. 2.
    Guo JT et al. (2000) Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections. J. Virol. 74:1495–505.CrossRefPubMedGoogle Scholar
  3. 3.
    Liang TJ, Heller T. (2004) Pathogenesis of hepatitis C-associated hepatocellular carcinoma. Gastroenterology 127: S62–71.CrossRefPubMedGoogle Scholar
  4. 4.
    Johnson RC. (1997) Hepatocellular carcinoma. Hepatogastroenterologgy 44:307–12.Google Scholar
  5. 5.
    Llovet JM, Burroughs A, Bruix J. (2003) Hepatocellular carcinoma. Lancet 362:1907–17.CrossRefPubMedGoogle Scholar
  6. 6.
    Bluteau O et al. (2002) Bi-allelic inactivation of TCF1 in hepatic adenomas. Nat. Genet. 32:312–5.CrossRefPubMedGoogle Scholar
  7. 7.
    Camma C, Giunta M, Andreone P, Craxi A. (2001) Interferon and prevention of hepatocellular carcinoma in viral cirrhosis: an evidence-based approach. J. Hepatol. 34:593–602.CrossRefPubMedGoogle Scholar
  8. 8.
    Kornek M et al. (2008) Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of pro-angiogenic and prometastatic factors in murine liver fibrosis. Liver Int. 28:509–18.CrossRefPubMedGoogle Scholar
  9. 9.
    Kuriyama S et al. (1999) Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver. Int. J. Cancer 80:471–6.CrossRefPubMedGoogle Scholar
  10. 10.
    Ferrara N, Kerbel RS. (2005) Angiogenesis as a therapeutic target. Nature 438:967–74.CrossRefPubMedGoogle Scholar
  11. 11.
    Kim SH et al.(2006) PEG conjugated VEGF siRNA for anti-angiogenic gene therapy. J. Control. Release 116:123–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Mulkeen AL et al. (2006) Short interfering RNA-mediated gene silencing of vascular endothelial growth factor: effects on cellular proliferation in colon cancer cells. Arch. Surg. 141:367–74.CrossRefPubMedGoogle Scholar
  13. 13.
    Jia RB et al. (2007) VEGF-targeted RNA interference suppresses angiogenesis and tumor growth of retinoblastoma. Ophthalmic Res. 39:108–15.CrossRefPubMedGoogle Scholar
  14. 14.
    Shen HL et al. (2007) Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells. Leuk. Res. 31:515–21.CrossRefPubMedGoogle Scholar
  15. 15.
    Kornek M et al. (2006) Combination of systemic thioacetamide (TAA) injections and ethanol feeding accelerates hepatic fibrosis in C3H/He mice and is associated with intrahepatic up regulation of MMP-2, VEGF and ICAM-1. J. Hepatol. 45: 370–6.CrossRefPubMedGoogle Scholar
  16. 16.
    Kornek M et al. (2007) Experimental orthotopic HCC growth is accelerated in hepatic fibrosis. J. Hepatol. 46:S82–3.CrossRefGoogle Scholar
  17. 17.
    Kornek M, Raskopf E, Tolba R, Becker U, Klöckner M, Sauerbruch T, Schmitz V. (2008) Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of proangiogenic and prometastatic factors in murine liver fibrosis. Liver Int. 28(4):509–18.CrossRefPubMedGoogle Scholar
  18. 18.
    Filleur S et al. (2003) SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63:3919–22.PubMedGoogle Scholar
  19. 19.
    Takei Y et al. (2004) A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res. 64:3365–70.CrossRefPubMedGoogle Scholar
  20. 20.
    Verma UN et al. (2003) Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin. Cancer Res. 9:1291–300.PubMedGoogle Scholar
  21. 21.
    Aigner A. (2006) Delivery systems for the direct application of siRNAs to Induce RNA interference (RNAi) in vivo. J. Biomed. Biotechnol. 2006: 71659.CrossRefPubMedGoogle Scholar
  22. 22.
    Ishak K et al. (1995) Histological grading and staging of chronic hepatitis. J. Hepatol. 22:696–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Judge AD et al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23: 457–62.CrossRefPubMedGoogle Scholar
  24. 24.
    Hornung V et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11:263–70.CrossRefPubMedGoogle Scholar
  25. 25.
    Schlee M, Hornung V, Hartmann G. (2006) siRNA and isRNA: two edges of one sword. Mol. Ther. 14:463–70.CrossRefPubMedGoogle Scholar
  26. 26.
    Khazanov E, Simberg D, Barenholz Y. (2006) Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice. J. Gene Med. 8:998–1007.CrossRefPubMedGoogle Scholar
  27. 27.
    Kim WJ et al. (2006) Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adeno’t takcarcinoma. Mol. Ther. 14:343–50.CrossRefPubMedGoogle Scholar
  28. 28.
    Niola F et al.(2006) Aplasmid-encoded VEGF siRNA reduces glioblastoma angiogenesis and its combination with interleukin-4 blocks tumor growth in a xenograft mouse model. Cancer Biol. Ther. 5:174–9.CrossRefPubMedGoogle Scholar
  29. 29.
    Raskopf E et al. (2005) Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model. Hepatology 41:1233–40.CrossRefPubMedGoogle Scholar
  30. 30.
    Schmitz V et al. (2006) Increased VEGF levels induced by anti-VEGF treatment are independent of tumor burden in colorectal carcinomas in mice. Gene Ther. 13:1198–205.CrossRefPubMedGoogle Scholar
  31. 31.
    Schmitz V et al. (2004) Establishment of an orthotopic tumour model for hepatocellular carcinoma and non-invasive in vivo tumour imaging by high resolution ultrasound in mice. J. Hepatol. 40:787–91.CrossRefPubMedGoogle Scholar
  32. 32.
    Raskopf E et al. (2007) Posttranscriptional VEGF inhibition reduces tumor growth in an experimental murine HCC model. J. Hepatol. 46:S151.CrossRefGoogle Scholar
  33. 33.
    Roberts N et al. (2006) Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res. 66:2650–7.CrossRefPubMedGoogle Scholar
  34. 34.
    Liu L et al. (2003) Poly(cationic lipid)-mediated in vivo gene delivery to mouse liver. Gene Ther. 10:180–7.CrossRefPubMedGoogle Scholar
  35. 35.
    Hamm S et al. (2007) Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo. Int. Immunol. 19:297–304.CrossRefPubMedGoogle Scholar
  36. 36.
    Meier A et al. (2007) MyD88-dependent immune activation mediated by human immunodeficiency virus type 1-encoded Toll-like receptor ligands. J. Virol. 81:8180–91.CrossRefPubMedGoogle Scholar
  37. 37.
    Hertzog PJ, Hwang SY, Kola I. (1994) Role of interferons in the regulation of cell proliferation, differentiation, and development. Mol. Reprod. Dev. 39:226–32.CrossRefPubMedGoogle Scholar
  38. 38.
    Hong YK et al. (2000) Efficient inhibition of in vivo human malignant glioma growth and angiogenesis by interferon-beta treatment at early stage of tumor development. Clin. Cancer Res. 6:3354–60.PubMedGoogle Scholar
  39. 39.
    Izawa JI et al. (2002) Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various antiangiogenic effects including endothelial cell apoptosis. Clin Cancer Res. 8:1258–70.PubMedGoogle Scholar
  40. 40.
    Cao G et al. (2001) Adenovirus-mediated interferonbeta gene therapy suppresses growth and metastasis of human prostate cancer in nude mice. Cancer Gene Ther. 8:497–505.CrossRefPubMedGoogle Scholar
  41. 41.
    Wang L et al. (2000) High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology 32:43–8.CrossRefPubMedGoogle Scholar
  42. 42.
    Ikeda K et al. (2000) Interferon beta prevents recurrence of hepatocellular carcinoma after complete resection or ablation of the primary tumor-A prospective randomized study of hepatitis C virus-related liver cancer. Hepatology 32:228–32.CrossRefPubMedGoogle Scholar
  43. 43.
    Ogasawara S et al. (2007) Growth inhibitory effects of IFN-beta on human liver cancer cells in vitro and in vivo. J. Interferon Cytokine Res. 27: 507–16.CrossRefPubMedGoogle Scholar
  44. 44.
    Shweiki D, Itin A, Soffer D, Keshet E. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359:843–5.CrossRefPubMedGoogle Scholar
  45. 45.
    Forsythe JA et al. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16: 4604–13.CrossRefPubMedGoogle Scholar
  46. 46.
    Jones DT, Harris AL. (2006) Identification of novel small-molecule inhibitors of hypoxiainducible factor-1 transactivation and DNA binding. Mol. Cancer Ther. 5:2193–202.CrossRefPubMedGoogle Scholar
  47. 47.
    Gillespie DL et al. (2007) Silencing of hypoxia inducible factor-1alpha by RNA interference attenuates human glioma cell growth in vivo. Clin. Cancer Res. 13:2441–8.CrossRefPubMedGoogle Scholar
  48. 48.
    Bocci G et al. (2004) Increased plasma vascular endothelial growth factor (VEGF) as a surrogate marker for optimal therapeutic dosing of VEGF receptor-2 monoclonal antibodies. Cancer Res. 64:6616–25.CrossRefPubMedGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2008

Authors and Affiliations

  • Miroslaw Kornek
    • 1
  • Veronika Lukacs-Kornek
    • 2
  • Andreas Limmer
    • 2
  • Esther Raskopf
    • 1
  • Ursula Becker
    • 1
  • Maren Klöckner
    • 1
  • Tilman Sauerbruch
    • 1
  • Volker Schmitz
    • 1
    • 3
  1. 1.Department of Internal Medicine IUniversity Hospital BonnBonnGermany
  2. 2.Institute of Molecular Medicine and Experimental Immunology (IMMEI)BonnGermany
  3. 3.Medizinische Klinik IBonnGermany

Personalised recommendations