Fluoro-Sorafenib (Regorafenib) effects on hepatoma cells: growth inhibition, quiescence, and recovery

Abstract

To evaluate the growth-inhibitory properties of the potent multi-kinase antagonist Regorafenib (Fluoro-Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, apoptosis, and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications.

Fig. 1. Hepatoma cell growth inhibition

A. Hep3B, PLC/PRF/5, HepG2 human hepatoma cell lines were cultured in presence of Regorafenib at various concentrations or in solvent alone. At 72 h, cells were harvested for MTT assay. The relative cell viability (%) was calculated as a percentage relative to the untreated control cells.: OD of treated cells / OD of control cells × 100 %. B. Hep3B cells were cultured in presence of 7.5 μM Regorafenib for either 0-24, 0-48 or 0-72 h and the medium was then changed to drug-free medium or for 0-96 h. All cells were harvested at 96 h and the viability was evaluated by the MTT assay.

Fig. 2. Regorafenib-mediated cell signaling

A. Regorafenib-mediated early cell signaling. A representative time-course of Regorafenib-induced phosphorylation of phospho-ERK, phospho-JNK and phospho-c-Jun in Hep3B cells treated continuously in culture with 7.5 μM Regorafenib for 15, 60 or 180 min. C, controls (solvent alone, drug-untreated cells). B. Same treatments as Fig. 2A, but cells were treated for and then harvested at 24 or 48 h. The β-actin, used as loading control, in treated and untreated cells show similar relative optical density at each time point. This reflects the accurate amount of loaded target proteins.

Fig. 3. Apoptosis and autophagy

A. FACS measurement for apoptosis after culture of Hep3B cells for 48 h with 2.5 (a), 5 (b), 7.5 (c) or 10 (d) μM Regorafenib. B and C. Western blotting of lysates from Hep3B cells treated with 7.5 μM Regorafenib for 24, 48 or 72 h and probed for apoptosis (B) and autophagy (C) markers. C, controls (solvent alone, drug-untreated cells). The β-actin protein was used as loading control.

Fig. 4. Reversibility of drug effects on cell growth

Hep3B cells were treated either with Regorafenib (4A) or Doxorubicin (4B) at different concentrations for 72 h in culture and the medium was then removed (t₀). The cells were washed with fresh medium and then cultured for the indicated further days in drug-free medium. The cells were harvested and the viability has been evaluated by the MTT assay.