Skip to main content
SpringerLink
Log in

Serum protein N-glycan alterations of diethylnitrosamine-induced hepatocellular carcinoma mice and their evolution after inhibition of the placental growth factor

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Placental growth factor (PlGF) inhibition produced promising results in reducing tumor burden in a diethylnitrosamine (DEN)-induced mouse model for hepatocellular carcinoma (HCC). The aim of this study was to non-invasively assess the improved histology by performing a serum glycomic analysis. To elucidate the molecular mechanism underlying the observed glycomic effects, we investigated the transcription and expression of E26 transformation-specific sequence 1 (Ets-1), a transcription factor essential for the glycomic and angiogenic changes in malignant transformation, including its different phosphorylated forms that result from activation of the MAP kinase and a Ca2+-dependent pathway. In addition, three Ets-1-dependent glycosyltransferase genes, Mgat4a, Mgat4b, and Mgat5, were also evaluated. HCC was induced in mice by weekly injections with DEN for 16, 20, 25, and 30 w. In the treatment study, mice were injected with DEN for 25 w and subsequently treated with PlGF antibodies (5D11D4) for 5 w. Finally, PlGF−/− mice were injected with DEN for 20, 25, and 30 w. Serum N-glycans were analyzed with DNA sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology. Maximum altered N-glycan phenotype was reached after 20 w of DEN-injections, i.e., when the first neoplastic lesions started to appear. 5D11D4-treatment improved the glycomic phenotype in that 7 of the 11 altered glycans tended to normalize. The PlGF−/− mice also showed a normalization trend, although not to the same extent of the treatment group. Number of Ets1, Mgat4a, Mgat4b, and Mgat5 transcripts increased considerably in DEN-injected mice, however, a non-significant decrease was observed after 5D11D4-treatment. On the protein level, 5D11D4-treatment had a prominent effect on the MAP kinase pathway with a significant p38 activation, yet independent of Ets-1 function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Nordenstedt H, White DL, El-Serag HB (2010) The changing pattern of epidemiology in Hepatocellular carcinoma. Dig Liver Dis 42(Suppl 3):S206–S214

    Article  PubMed  Google Scholar 

  2. Sherman M (2005) Hepatocellular carcinoma: epidemiology, risk factors, and screening. Semin Liver Dis 25:143–154

    Article  PubMed  Google Scholar 

  3. Piccinino F, Sagnelli E, Pasquale G, Giusti G (1986) Complications following percutaneous liver biopsy. A multicentre retrospective study on 68,276 biopsies. J Hepatol 2:165–173

    Article  PubMed  CAS  Google Scholar 

  4. Callewaert N, Van Vlierberghe H, Van Hecke A, Laroy W, Delanghe J, Contreras R (2004) Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat Med 10:429–434

    Article  PubMed  CAS  Google Scholar 

  5. Liu XE, Desmyter L, Gao CF et al (2007) N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus. Hepatology 46:1426–1435

    Article  PubMed  CAS  Google Scholar 

  6. Xu J, Yun X, Jiang J et al (2010) Hepatitis B virus X protein blunts senescence-like growth arrest of human hepatocellular carcinoma by reducing Notch1 cleavage. Hepatology 52:142–154

    Article  PubMed  CAS  Google Scholar 

  7. Shim JK, Lee YC, Chung TH, Kim CH (2004) Elevated expression of bisecting N-acetylglucosaminyltransferase-III gene in a human fetal hepatocyte cell line by hepatitis B virus. J Gastroenterol Hepatol 19:1374–1387

    Article  PubMed  CAS  Google Scholar 

  8. Yanagi M, Aoyagi Y, Suda T, Mita Y, Asakura H (2001) N-Acetylglucosaminyltransferase V as a possible aid for the evaluation of tumor invasiveness in patients with hepatocellular carcinoma. J Gastroenterol Hepatol 16:1282–1289

    Article  PubMed  CAS  Google Scholar 

  9. Goldman R, Ressom HW, Varghese RS et al (2009) Detection of hepatocellular carcinoma using glycomic analysis. Clin Cancer Res 15:1808–1813

    Article  PubMed  CAS  Google Scholar 

  10. Kang R, Saito H, Ihara Y et al (1996) Transcriptional regulation of the N-acetylglucosaminyltransferase V gene in human bile duct carcinoma cells (HuCC-T1) is mediated by Ets-1. J Biol Chem 271:26706–26712

    Article  PubMed  CAS  Google Scholar 

  11. Ko J, Miyoshi E, Noda K et al (1999) Regulation of the GnT-V promoter by transcription Ets-1 in various cancer cell lines. J Biol Chem 274:22941–22948

    Article  PubMed  CAS  Google Scholar 

  12. Sato Y (2001) Role of ETS family transcription factors in vascular development and angiogenesis. Cell Struct Funct 26:19–24

    Article  PubMed  CAS  Google Scholar 

  13. Wernert N, Raes MB, Lassalle P et al (1992) c-ets1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans. Am J Pathol 140:119–127

    PubMed  CAS  Google Scholar 

  14. Yang B, Hauser C, Henkel G et al (1996) Ras-mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c-Ets-1 and c-Ets-2. Moll Cell Biol 16:538–547

    CAS  Google Scholar 

  15. Cowley D, Graves B (2000) Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition. Genes Dev 14:366–376

    PubMed  CAS  Google Scholar 

  16. Pufall M, Lee G, Nelson M et al (2005) Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region. Science 309:142–145

    Article  PubMed  CAS  Google Scholar 

  17. Maglione D, Guerriero V, Viglietto G, Dellibovi P, Persico M (1991) Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci USA 88:9267–9271

    Article  PubMed  CAS  Google Scholar 

  18. Fischer C, Jonckx B, Mazzone M et al (2007) Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 131:463–475

    Article  PubMed  CAS  Google Scholar 

  19. Van de Veire S, Stalmans I, Heindryckx F et al (2010) Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease. Cell 141:178–190

    Article  PubMed  Google Scholar 

  20. Tang Z, Varghese R, Bekesova S et al (2010) Identification of N-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data. J Proteome Res 9:104–112

    Article  PubMed  CAS  Google Scholar 

  21. Heindryckx F, Mertens K, Charette N et al (2010) Kinetics of angiogenic changes in a new mouse model for hepatocellular carcinoma. Mol Cancer 9:219–232

    Article  PubMed  Google Scholar 

  22. Laroy W, Contreras R, Callewaert N (2006) Glycome mapping on DNA sequencing equipment. Nat Protoc 1:397–405

    Article  PubMed  CAS  Google Scholar 

  23. Blomme B, Van Steenkiste C, Callewaert N, Van Vlierberghe H (2011) Alterations of serum protein N-glycosylation in two models of chronic liver disease are hepatocyte and not B cell driven. Am J Physiol Gastrointest Liver Physiol 300:G833–G842

    Article  PubMed  CAS  Google Scholar 

  24. Blomme B, Van Steenkiste C, Vanhuysse J, Colle I, Callewaert N, Van Vlierberghe H (2010) Impact of elevation of total bilirubin level and etiology of the liver disease on serum N-glycosylation patterns in mice and humans. Am J Physiol Gastrointest Liver Physiol 298:G615–G624

    Article  PubMed  CAS  Google Scholar 

  25. Klein A, Carre Y, Louvet A, Michalski JC, Morelle W (2010) Immunoglobulins are the major glycoproteins involved in the modifications of total serum N-glycome in cirrhotic patients. Proteomics Clin Appl 4:379–393

    Article  PubMed  CAS  Google Scholar 

  26. Vanderschaeghe D, Laroy W, Sablon E et al (2009) GlycoFibroTest is a highly performant liver fibrosis biomarker derived from DNA sequencer-based serum protein glycomics. Mol Cell Proteomics 8:986–994

    Article  PubMed  CAS  Google Scholar 

  27. Liu XE, Dewaele S, Vanhooren V et al (2010) Alteration of N-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a non-invasive monitoring tool for liver cancer. Liver Int 30:1221–1228

    Article  PubMed  CAS  Google Scholar 

  28. Ito Y, Miyoshi E, Takeda T et al (2000) Expression and possible role of ets-1 in hepatocellular carcinoma. Am J Clin Pathol 114:719–725

    Article  PubMed  CAS  Google Scholar 

  29. Vanderschaeghe D, Szekrényes A, Wenz C et al (2010) High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems: towards clinical implication of the GlycoHepatoTest. Anal Chem 82:7408–7415

    Article  PubMed  CAS  Google Scholar 

  30. Takamatsu S, Antonopoulos A, Ohtsubo K et al (2010) Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice. Glycobiology 20:485–497

    Article  PubMed  CAS  Google Scholar 

  31. Mendoza R, Moody E, Enriquez M, Mejia S, Thordarson G (2011) Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase. J Endocrinol 208:11–19

    Article  PubMed  CAS  Google Scholar 

  32. Hsu Y, Meng X, Ou L, Ip M (2010) Activation of the AMP-activated protein kinase-p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells. Cell Signal 22:590–599

    Article  PubMed  CAS  Google Scholar 

  33. Hui L, Bakiri L, Stepniak E, Wagner E (2007) P38 alpha: a suppressor of cell proliferation and tumorigenesis. Cell Cycle 6:2429–2433

    Article  PubMed  CAS  Google Scholar 

  34. Hui L, Bakiri L, Mairhorfer A et al (2007) P38 alpha suppresses normal and cancer cell proliferation by antagonizing the JNK-c-Jun pathway. Nat Genet 39:741–749

    Article  PubMed  CAS  Google Scholar 

  35. McMullen M, Bryant P, Glembotski C, Vincent P, Pumiglia K (2005) Activation of p38 has opposing effects on the proliferation and migration of endothelial cells. J Biol Chem 280:20995–21003

    Article  PubMed  CAS  Google Scholar 

  36. Issbrücker K, Marti H, Hippenstiel S (2003) p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular permeability. FASEB J 17:262–264

    PubMed  Google Scholar 

  37. Luttun A, Brusselmans K, Fukao H et al (2002) Loss of placental growth factor protects mice against vascular permeability in pathological conditions. Biochem Biophys Res Commun 295:428–434

    Article  PubMed  CAS  Google Scholar 

  38. Vetter M, Blumenthal S, Lindemann R (2005) Ets1 is an effector of protein kinase Cα in cancer cells. Oncogene 24:650–661

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

BB receives a scholarship GOA BOFF07/GOA/017 of the University Ghent Research Fund (BOF). HVV is Senior Clinical Investigator of the Research Foundation (FWO). FH is funded by a FWO grant (Aspirant FWO, FWO09/ASP/161).

Conflict of interest

JM Stassen is a shareholder and Head of pre-clinical R&D of ThromboGenics NV and named in a patent related to data reported in this article which may result in payment of royalties. The other authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Hepatology and Gastroenterology, Ghent University Hospital, 9000, Ghent, Belgium

    Bram Blomme, Femke Heindryckx, Anja Geerts, Isabelle Colle & Hans Van Vlierberghe

  2. ThromboGenics NV, Leuven, Belgium

    Jean Marie Stassen

Authors
  1. Bram Blomme
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Femke Heindryckx
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean Marie Stassen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anja Geerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Isabelle Colle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans Van Vlierberghe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bram Blomme.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blomme, B., Heindryckx, F., Stassen, J.M. et al. Serum protein N-glycan alterations of diethylnitrosamine-induced hepatocellular carcinoma mice and their evolution after inhibition of the placental growth factor. Mol Cell Biochem 372, 199–210 (2013). https://doi.org/10.1007/s11010-012-1461-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-012-1461-1

Keywords

Search

Navigation