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Reversibility of regorafenib effects in hepatocellular carcinoma cells

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Abstract

Purpose

Multikinase growth inhibitors inhibit their target kinases with varying potency. Patients often require lower doses or therapy breaks due to drug toxicities. To evaluate the effects of drug withdrawal on hepatocellular carcinoma cells after incubation with growth-inhibitory concentrations of regorafenib, cell growth, migration and invasion, and signaling were examined.

Methods

Cell proliferation, motility, and invasion were analyzed by MTT, wound healing, and invasion assays, respectively, and MAPK pathway protein markers were analyzed by Western blot.

Results

After regorafenib removal, cell growth, migration, and invasion recovered. Repeated drug exposure resulted in changes in cell growth patterns. Recovery could be blocked by sub-growth-inhibitory concentrations of either doxorubicin or vitamin K1. Recovery of growth was associated with increased phospho-JNK, phospho-p38, and phospho-STAT3 levels. The recovery of growth, migration, and signaling were blocked by a JNK inhibitor.

Conclusions

Removal of regorafenib from growth-inhibited cells resulted in a JNK-dependent recovery of growth and migration.

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Acknowledgments

Grant support: This work was supported (in part) by NIH Grant [# 82723 (BIC)].

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Authors and Affiliations

  1. Laboratory of Biochemistry, National Institute for Digestive Diseases, IRCCS “Saverio de Bellis”, Via Turi, 27, 70013, Castellana Grotte, BA, Italy

    Rosalba D’Alessandro, Maria G. Refolo, Catia Lippolis, Caterina Messa, Aldo Cavallini & Brian I. Carr

  2. Clinical Pathology Laboratory, National Institute for Digestive Diseases, IRCCS “Saverio de Bellis”, Via Turi, 27, 70013, Castellana Grotte, BA, Italy

    Antonio Di Carlo

  3. Department of Pathological Anatomy, Faculty of Medicine, University of Bari, Giulio Cesare Square, 70100, Bari, BA, Italy

    Roberta Rossi & Leonardo Resta

Authors
  1. Rosalba D’Alessandro
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  2. Maria G. Refolo
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  3. Catia Lippolis
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  4. Caterina Messa
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  8. Antonio Di Carlo
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  9. Brian I. Carr
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Correspondence to Brian I. Carr.

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280_2013_2269_MOESM1_ESM.tif

Fig. 1S. Western blot of apoptosis and autophagy markers and electron microscopy analysis. A. Apoptosis and autophagy in Hep3B cells from T0 to T4. Up-regulation of anti-apoptotic proteins (pBcl-2 and Bcl-xL) and decrease in Beclin-1 and LC3 II as autophagy markers. B. Representative electron micrographs of Hep3B cells during recovery. 1 – Cells treated with regorafenib 5 µM for 72h (T0). White arrow shows large clumps of heterochromatin and black arrow typical autophagosome. (Bar = 2μm; magnification x4,400). 2 – Cells during reversibility (T3). The autophagy or apoptotic structures are not present in the cytoplasm. (Bar = 2μm; magnification x4,400 ). 3 – High magnification of autophagosome of image (1) (black arrow) (Bar = 0.5μm; magnification x11,000) (TIFF 540 kb)

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D’Alessandro, R., Refolo, M.G., Lippolis, C. et al. Reversibility of regorafenib effects in hepatocellular carcinoma cells. Cancer Chemother Pharmacol 72, 869–877 (2013). https://doi.org/10.1007/s00280-013-2269-8

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  • DOI: https://doi.org/10.1007/s00280-013-2269-8

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