Ponatinib Inhibits Proliferation and Induces Apoptosis of Liver Cancer Cells, but Its Efficacy Is Compromised by Its Activation on PDK1/Akt/mTOR Signaling

Abstract

Ponatinib is a multi-target protein tyrosine kinase inhibitor, and its effects on hepatocellular carcinoma cells have not been previously explored. In the present study, we investigated its effects on hepatocellular carcinoma cell growth and the underlying mechanisms. Toward SK-Hep-1 and SNU-423 cells, ponatinib induces apoptosis by upregulation of cleaved caspase-3 and -7 and promotes cell cycle arrest in the G1 phase by inhibiting CDK4/6/CyclinD1 complex and phosphorylation of retinoblastoma protein. It inhibits the growth-stimulating mitogen-activated protein (MAP) kinase pathway, the phosphorylation of Src on both negative and positive regulation sites, and Jak2 and Stat3 phosphorylation. Surprisingly, it also activates the PDK1, the protein kinase B (Akt), and the mechanistic target of rapamycin (mTOR) signaling pathway. Blocking mTOR signaling strongly sensitizes cells to inhibition by ponatinib and makes ponatinib a much more potent inhibitor of hepatocellular carcinoma cell proliferation. These findings demonstrate that ponatinib exerts both positive and negative effects on hepatocellular cell proliferation, and eliminating its growth-stimulating effects by drug combination or potentially by chemical medication can significantly improve its efficacy as an anti-cancer drug.

Keywords: PDK1/Akt/mTOR signaling; apoptosis; liver cancer cells; ponatinib; proliferation.

Figure 1

Efficacy of individual PTK inhibitors against different hepatocellular carcinoma (HCC) cell lines and cytotoxicity of ponatinib. (A) Cell proliferation was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. HCC cell lines including SK-Hep-1 and SNU-423 cells were seeded in 96-well plates and then treated with different concentrations of drugs for 72 h. The relative cell viability was calculated and shown. (B) Cell proliferation inhibition was observed by microscopy (×100). SK-Hep-1 and SNU-423 cells were plated in 24-well plates and exposed to indicated doses of ponatinib for 36 h. (C) SK-Hep-1 and SNU-423 cells were plated in six-well plates (500 cells/well) and treated with various concentrations of ponatinib for 10 days, then the clonogenic ability was measured and shown. The bars indicate the colony formation ability of different groups. Each experiment was carried out three times. (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 2

Ponatinib induced cell apoptosis in SK-Hep-1 and SNU-423 cells in a concentration-dependent manner. (A) SK-Hep-1 and SNU-423 cells were stained with Hoechst 33342 observed on fluorescence microscopy (×200). (B) Flow cytometry analysis of apoptosis (data from three independent experiments). (C) Western blot analysis of cleaved caspase-3, cleaved caspase-7, Bax, Bcl-2, and β-Tubulin (control) in treated cells.

Figure 3

Effect of Ponatinib on the percentage of cells in the G1 phase and the expression of G1 phase-related proteins. (A) SK-Hep-1 and SNU-423 cells were treated with ponatinib, and cell cycle distribution was analyzed by flow cytometry. The percentages of cells in cell cycle phases were calculated from three independent experiments. (B) Western blot analysis of Rb/p-Rb, CDK4, CDK6, Cyclin D1, CDK2, Cyclin E1, and β-Tubulin (control) in the SK-Hep-1 and SNU-423 cells treated with ponatinib. (C) The effect of ponatinib on the concentration of Rad51 in SK-Hep-1 and SNU-423 cells.

Figure 4

Effect of ponatinib on the phosphorylation of signaling proteins. (A) Effect of ponatinib on the phosphorylation of Src on Tyr416 and Tyr527. (B–E) Effect of ponatinib on the phosphorylation of Mek, Erk, JNK, c-Jun, p38, Jak2, and Stat3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.

Figure 5

Effect of ponatinib on the protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling pathway. (A) Effects of ponatinib on the protein concentration and phosphorylation of PI 3-kinase, PDK1, Akt, and mTOR. (B) Time course of ponatinib (1 μM) effect on the phosphorylation of Akt and mTOR. (C) Effect of ponatinib on SK-Hep-1 cell viability in the presence and absence of mTOR inhibitors, temsirolimus and rapamycin. (D) Effect of ponatinib on SNU-423 cell viability in the presence and absence of mTOR inhibitors, temsirolimus and rapamycin. (E) Effect of ponatinib on SK-Hep-1 cell viability in the presence and absence of an Akt inhibitor, MK-2206. (F) Effect of ponatinib on SNU-423 cell viability in the presence and absence of an Akt inhibitor, MK-2206.

Figure 6

Effect of ponatinib on PTEN. (A) Effect of ponatinib on the phosphorylation of PTEN on Ser380/Thr382/383. For the Western blot analysis, SK-Hep-1 and SNU-423 cells were treated with ponatinib at indicated concentrations for 36 h. (B) Time course of ponatinib (1 μM) effect on PTEN in SK-Hep-1 and SNU-423 cells.