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Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation

  • Original Article - Cancer Research
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Purpose

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR1 and PAR4 are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms.

Methods

Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H2DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate.

Results

Thrombin, the PAR1 selective agonist peptide TFLLRN-NH2 (PAR1-AP), and the PAR4 selective agonist peptide, AYPGKF-NH2 (PAR4-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR1-AP and the PAR4-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR1- and PAR4-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-l-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR1-AP and the PAR4-AP, but had no effect on PAR1/4-mediated activation of Met and PDGFR in Hep3B cells.

Conclusions

Collectively, our data indicate that PAR1 and PAR4 activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.

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Abbreviations

DCF:

Dichlorofluorescein

EGFR:

Epidermal growth factor receptor

FGFR:

Fibroblast growth factor receptor

GPCR:

G protein-coupled receptor

HCC:

Hepatocellular carcinoma

IGFR:

Insulin-like growth factor receptor

Met:

Hepatocyte-growth factor receptor

PAR:

Proteinase-activated receptor

PDGFR:

Platelet-derived growth fcator receptor

PLC:

Phospholipase C

PTP:

Protein tyrosine phosphatase

ROS:

Reactive oxygen species

RTK:

Receptor tyrosine kinase

VEGFR:

Vascular endothelial growth factor receptor

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Acknowledgments

The authors would like to thank Beate Schulze, Elke Oswald (Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Jena University Hospital and Frank Steiniger (Electron Microscopy Center, Jena University Hospital) for the excellent technical assistance. This work was supported by the Deutsche Krebshilfe (Grant 108809, to R.K. and U.S.).

Conflict of interest

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Department of General, Visceral and Vascular Surgery, Jena University Hospital, Erlanger Allee 101, 07747, Jena, Germany

    Franziska Mußbach, Utz Settmacher & Roland Kaufmann

  2. Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Jena University Hospital, Drackendorfer Str. 1, 07747, Jena, Germany

    Franziska Mußbach & Roland Kaufmann

  3. Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CharitéCrossOver (CCO), Berlin, Germany

    Petra Henklein

  4. Electron Microscopy Center, Jena University Hospital, Jena, Germany

    Martin Westermann

  5. Center for Molecular Biomedicine, Institute of Molecular Cell Biology, Jena University Hospital, Jena, Germany

    Frank-D. Böhmer

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  1. Franziska Mußbach
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  2. Petra Henklein
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Correspondence to Roland Kaufmann.

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Mußbach, F., Henklein, P., Westermann, M. et al. Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation. J Cancer Res Clin Oncol 141, 813–825 (2015). https://doi.org/10.1007/s00432-014-1863-4

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