The Eltrombopag antitumor effect on hepatocellular carcinoma

Abstract

Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia.

Figure 1

Effect of eltrombopag (EP) on human HCC cell lines. Cell lines were treated with 0–100 μg/ml EP. The inhibited cell proliferation rates are shown for (A) Huh7, (B) HepG2 and (C) Hep3B, respectively. ^*P<0.05 compared to 0 μg/ml EP. The values indicate ratio compared to 0 μg/ml EP as 100% control.

Figure 2

Preloading cells with iron resulted in a rescue from the antiproliferative effects of eltrombopag. Huh7 cells were untreated or treated with 0.1–100 μg/ml of EP 72 h preloaded or not with 500 μg/ml of ferric ammonium citrate (FAC) for 24 h. Cell viability was measured with a CCK-8 assay performed at 72 h. Comparison with the data without iron preloading. (A) Huh7, (B) HepG2 and (C) Hep3B. ^*P<0.05 compared to each dose EP not preloaded of FAC. The values indicate a ratio compared to 0 μg/ml EP as 100% control.

Figure 3

Effect of eltrombopag on cell cycle progression in Huh7 cells. (A) Cells were treated with EP (0 and 10 μg/ml) for 72 h. DNA was stained with propidium iodide, and flow cytometric analysis of cell phase distribution was performed using a Muse™ cell analyzer. (B) Cells were treated with EP (0, 1 and 10 μg/ml) for 72 h. Protein expression was determined by western blot analysis using anti-cyclin D1, anti-p21 and anti-GAPDH antibodies. SDS-PAGE was performed on acrylamide gels.

Figure 4

Comparison of the antitumor effect of DFO and eltrombopag (EP). Huh7 cells were treated with 0.1–100 μg/ml of DFO for 72 h, or not treated. Cell viability was measured by a CCK-8 assay performed at 72 h. ^*P<0.05 compared to the same concentration of EP. The values indicate a ratio compared to 0 μg/ml DFO as 100% control.

Figure 5

Combined effects of eltrombopag (EP) and sorafenib on HCC. (A) Huh7 cells were treated with 0.1–100 μg/ml of EP and sorafenib (0 and 4 μM), or not treated. ^*P<0.05 compared to 0 μg/ml EP and 4 μM sorafenib. The values indicate a ratio compared to 0 μg/ml EP and 4 μM sorafenib as 100% control. (B) Combination index values were determined using the method described by Chou (19); CI=1, cumulative effect; CI<1, synergistic effect; and CI>1, antagonistic effect.

Figure 6

The schematic chart of the EP function for the regeneration and carcinoma in human liver. Platelets suppress hepatic fibrosis and promote liver regeneration. EP could be developed into a novel treatment for LC by increment of platelet counts in clinical settings. EP also exhibited antitumor activity in HCC by eliciting cell cycle arrest through iron chelation compared to without TPO-R.