Fragile histidine triad (FHIT) suppresses proliferation and promotes apoptosis in cholangiocarcinoma cells by blocking PI3K-Akt pathway

Abstract

Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.

Figure 1

The expression of FHIT in QBC939 cells after transfection. (a) The expression of FHIT was assessed in each group after transfection using quantitative real-time RT-PCR. (b) Western blotting for FHIT protein expression. (c) Quantification of FHIT protein expression in each group presented in bar graphs as fold-increase. **P < 0.01.

Figure 2

The levels of p-Akt were assessed by western blotting. (a) Western blotting for p-Akt. (b) Quantification of p-Akt protein levels in each group, presented in bar graphs as fold-increase. *P < 0.05 versus NC/FHIT; **P < 0.001 versus NC/LY294002.

Figure 3

The expression of survivin, Bcl-2, and caspase-3 in QBC939 cells in each group after transfection or treatment with LY294002. (a) The mRNA expression of survivin, Bcl-2, and caspase-3 in each group, as assessed by RT-PCR. (b) Western blotting for survivin, Bcl-2, and caspase-3. (c) Quantification of the expression levels of survivin, Bcl-2, and caspase-3 in each group, presented in bar graphs as fold-increase. *P < 0.05, **P < 0.01.

Figure 4

Effect of FHIT overexpression and LY294002 treatment on cell proliferation. OD (optical density) was used to assess cell proliferation at 0, 24, 48, and 72 h after treatment with LY294002 or transfection.

Figure 5

Apoptosis in each group was assessed after transfection or treatment with LY294002. Apoptosis was assessed after 48 h treatment with 20 μmol LY294002. **P < 0.01.