Effects of low concentrations of regorafenib and sorafenib on human HCC cell AFP, migration, invasion, and growth in vitro

Abstract

Sorafenib was shown in clinical trial to enhance survival in hepatocellular carcinoma (HCC) patients, but with minimal tumor shrinkage. To correlate several indices of HCC growth at various drug concentrations, HCC cells were grown in various low concentrations of two multikinase inhibitors, regorafenib (Stivarga) and sorafenib (Nexavar) and their effects were examined on alpha-fetoprotein (AFP), cell growth, migration, and invasion. In two AFP positive human HCC cell lines, AFP was inhibited at 0.1-1 µM drug concentrations. Cell migration and invasion were also inhibited at similar low drug concentrations. However, 10-fold higher drug concentrations were required to inhibit cell growth in both AFP positive and negative cells. To investigate this concentration discrepancy of effects, cells were then grown for prolonged times and sub-cultured in low drug concentrations and then their growth was re-tested. The growth in these drug-exposed cells was found to be slower than cells without prior drug exposure and they were also more sensitive to subsequent drug challenge. Evidence was also found for changes in cell signaling pathways in these slow-growth cells. Low multikinase inhibitor concentrations thus modulate several aspects of HCC cell biology.

Fig. 1. Effects of Regorafenib and Sorafenib on cell proliferation

Growth curves for three different human HCC cell lines, PLC/PRF/5, HepG2 and Hep3B, treated with drug for 24, 48 and 72 hours.

Fig. 2. AFP changes after Regorafenib or Sorafenib exposure

A: AFP levels in the cell culture medium of PLC/PRF/5 and HepG2 cell lines and the number of viable cells after treatment with different drug concentrations. B: Western blotting and real-time PCR analysis of cellular AFP in relation to drug concentrations. The experiments were repeated three times. # Average of changes in protein band densities using arbitrary control values as unity. The relative AFP mRNA expression (mean ± SD) is expressed as AFP/β-actin ratio. C = control, vehicle treated cells.

Fig. 3. Effects of Regorafenib or Sorafenib on cell migration and invasion

A: Migration assay. PLC/PRF/5 cells were treated with the drugs and microscopically analyzed at the time of the scratch (T0) and after 48 and 72 hours. Original photographs and quantitative analysis of the cell-free scratch path areas, each performed in triplicate, are shown. B: Migration and Invasion assays quantization. Invasion assay: Invading PLC/PRF/5 cells were treated with different drug concentrations. Invasion was calculated as a percentage of the invading drug-treated cells compared to drug-untreated control cells.

Fig. 4. Cell growth after 4 weeks of exposure to 1 µM Regorafenib or Sorafenib

A: Cell growth after release from drug exposure (0.1µM for HepG2 cells and 1µM for PLC/PRF/5 and Hep3B cells) for 4 weeks. B: Cell growth after release from Regorafenib 0.1 or 1µM as in A, and subsequent challenge with various Regorafenib concentrations (ii), compared with the same challenge to non-pretreated cells (i).

Fig. 5. Signaling in cells treated with prolonged Regorafenib or Sorafenib

Western blotting analysis for phosphorylated ERK (P-ERK) and phosphorylated MEK (P-MEK) (compared with ERK and MEK) in Hep3B and PLC/PRF/5 cells after exposure to either 1µM Regorafenib or Sorafenib for 1 or 2 weeks (1W; 2W).