A ligand for peroxisome proliferator activated receptor gamma inhibits cell growth and induces apoptosis in human liver cancer cells

Abstract

Background and aims: Induction of apoptosis of cancer cells through ligands of nuclear hormone receptors (NHRs) is a new approach in cancer therapy. Recently, one of the NHRs, peroxisome proliferator activated receptor gamma (PPARgamma), has been shown to influence cell growth in certain cancer cells although its effect on hepatocellular carcinoma (HCC) has not been analysed.

Methods: Experiments were conducted using three human liver cancer cell lines, PLC/PRF/5, Hep G2 and HuH-7, in vitro. These cells were exposed to troglitazone, a synthetic ligand for PPARgamma, and the effects on cell growth were analysed.

Results: Expression of PPARgamma mRNA was detected in all three liver cancer cell lines. Activation of PPARgamma by troglitazone caused a marked growth inhibition in a dose dependent manner in three hepatoma cell lines. The DNA fragmentation ELISA assay and Hoechst 33258 staining revealed that the growth inhibitory effect by adding troglitazone was due to apoptosis of PLC/PRF/5, which strongly expressed PPARgamma. Troglitazone also induced activation of the cell death protease, caspase 3, but not caspase 8, in PLC/PRF/5 cells. However, expression levels of antiapoptotic factor bcl-2 and apoptosis inducing factor bax were not affected.

Conclusion: Our study showed that PPARgamma was expressed in human liver cancer cells and that the ligand for PPARgamma, troglitazone, inhibited the growth of these cells by inducing apoptosis through caspase 3 activation, indicating that troglitazone could be potentially useful as an apoptosis inducer for the treatment of HCC.

Figure 1

Northern blot analysis revealed peroxisome proliferator activated receptor γ (PPARγ) expression in all three liver cancer cell lines (PLC/PRF/5, Hep G2, and HuH-7). The expression level of PPARγ mRNA was higher in PLC/PRF/5 than in Hep G2 or HuH-7.

Figure 2

(A) Effect of troglitazone on the growth of liver cancer cell lines. The growth of all three liver cancer cell lines was inhibited by troglitazone treatment in a dose dependent manner. Data are presented as per cent growth relative to untreated cells (120 hours). (B–D) Time course of inhibition of cell growth by troglitazone. Treatment with 10 μM of troglitazone resulted in significant growth inhibition of all three cell lines (PLC/PRF/5 (B), Hep G2 (C), and HuH-7 (D)) up to 120 hours.

Figure 3

DNA fragmentation by ELISA assay, as measured by absorbance (OD 450 values). Culture of PLC/PRF/5 for 72 hours in the presence of troglitazone resulted in dose dependent DNA fragmentation.

Figure 4

Detection of apoptosis of PLC/PRF/5 cells by Hoechst 33258 staining. PLC/PRF/5 treated with 20 μM of troglitazone for 72 hours showed morphological features of apoptosis. In contrast, untreated cells maintained normal chromatin patterns and cell size (original magnification ×200).

Figure 5

Effect of troglitazone on caspases 3 (A) and 8 (B) in PLC/PRF/5 cells. PLC/PRF/5 cells were treated for 48 hours with 5 or 10 μM of troglitazone. The activity of caspase 3, but not that of caspase 8, was significantly increased in response to troglitazone in a dose dependent manner. **p<0.01.

Figure 6

Effect of troglitazone on expression of bcl-2 and bax. Western blot analysis demonstrated that expression levels of bcl-2 and bax in PLC/PRF/5 were not changed by treatment with troglitazone for 96 hours.