Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells

Abstract

Background: Involvement of peroxisome proliferator activated receptor gamma (PPARgamma) in the growth response of colon cancer cells has been suggested.

Aims: To investigate the characteristics of PPARgamma induced apoptosis in colon cancer cells.

Methods: The effects of ligands for each of the PPAR subtypes (alpha, delta, and gamma) on DNA synthesis and cell viability were examined in HT-29 colon cancer cells. Modulation of apoptosis related gene expression by PPARgamma ligands was screened with cDNA arrays, and the results were confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Results: PPARalpha, PPARdelta, and PPARgamma were all expressed in HT-29 cells. PPARgamma ligands, 15-deoxy-delta(12,)(14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of HT-29 cells whereas ligands for PPARalpha and PPARdelta had no significant effects. Both 15d-PGJ2 and TGZ induced HT-29 cell death in a dose dependent manner which was associated with an increase in fragmented DNA and was sensitive to a caspase inhibitor. Among several genes selected by cDNA array screening, quantitative RT-PCR analysis confirmed downregulation of c-myc expression and upregulation of c-jun and gadd153 expression by 15d-PGJ2 and TGZ. PPARgamma induced apoptosis was antagonised by the presence of serum in the culture medium, and interaction between PPARgamma signalling and cell survival signalling through the phosphatidylinositol 3-kinase pathway was suggested.

Conclusions: As c-myc is an important target gene of the adenomatous polyposis coli (APC)/beta-catenin and/or APC/gamma-catenin pathway, activation of PPARgamma signalling appears to compensate for deregulated c-myc expression caused by mutated APC. The present results suggest the potential usefulness of PPARgamma ligands for chemoprevention and treatment of colon cancers.

Figure 1

Western blot analysis showing expression of peroxisome proliferator activated receptor α (PPARα), δ (PPARδ), and γ (PPARγ) proteins in HT-29 cells.

Figure 2

Effects of peroxisome proliferator activated receptor (PPAR) ligands on DNA synthesis (bromodeoxyuridine (BrdU) incorporation) of HT-29 cells (30 hours of incubation) (n=6–12). Experiments were performed in the presence of 10% fetal bovine serum. (A) Wy-14643 (PPARα ligand) (1–100 μM), (B) carbaprostacyclin (cPGI, PPARδ ligand) (1–100 μM), (C) 15-deoxy-δ- prostaglandin J₂ (15d-PGJ₂, PPARγ ligand) (1–100 μM), and (D) troglitazone (TGZ, PPARγ ligand) (1–100 μM). **p<0.01 versus control.

Figure 3

Effects of different concentrations of 15-deoxy-δ- prostaglandin J₂ (15d-PGJ₂ 1–100 μM) and troglitazone (TGZ 1–100 μM) on HT-29 cell viability (26 hour incubation) in the presence (A, B) and absence (C, D) of 10% fetal bovine serum (n=8–12). **p<0.01 versus control.

Figure 4

Characterisation of peroxisome proliferator activated receptor γ (PPARγ) ligand induced HT-29 cell death. Experiments were performed in the absence of fetal bovine serum. (A) Effects of 18 hours of treatment with 15-deoxy-δ- prostaglandin J₂ (15d-PGJ₂ 10 μM) or troglitazone (TGZ 20 μM) on the amount of fragmented DNA (n=6–8). **p<0.01 versus control. (B) Effect of a caspase inhibitor (CI), Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fmk (25 μM), on TGZ (30 μM) induced HT-29 cell death (n=6–8). HT-29 cells were preincubated with 25 μM CI for two hours and further incubated with 20 μM TGZ for 24 hours in the continued presence of the caspase inhibitor. p<0.01 TGZ versus CI/TGZ. (C) Effect of cycloheximide (CHX 25 μM) on TGZ (20 μM) induced HT-29 cell death (n=6–8). Cells were preincubated with 25 μM CHX for two hours and further incubated with 20 μM TGZ for 24 hours in the continued presence of CHX. p<0.01 TGZ versus CHX/TGZ.

Figure 5

Effects of 9-cis-retinoic acid (9c-RA) and all-trans retinoic acid (AT-RA) on HT-29 cell viability. Experiments were performed in the absence of fetal bovine serum. Cells were incubated with 9c-RA (10 μM) or AT-RA (10 μM) in combination with 15-deoxy-δ- prostaglandin J₂ (15d-PGJ₂ 10 μM) or troglitazone (TGZ 20 μM) for 26 hours (n=6–8).

Figure 6

Representative standard curves for quantitative reverse transcription-polymerase chain reaction. (A) For β-actin, the threshold cycles (Ct)—that is, the polymerase chain reaction cycle number at which the fluorescence signal reached above baseline—was −3.36 log (starting copy number) + 38.07, r²=0.996. (B) For c-myc, Ct=−3.38 log (starting copy number) + 38.63, r²=0.997. (C) For c-jun, Ct=−3.45 log (starting copy number) + 39.44, r²=0.997. (D) For gadd153, Ct=−3.42 log (starting copy number) + 39.69, r²=0.999.

Figure 7

Quantitative reverse transcription-polymerase chain reaction analysis showing the effects of eight hours of treatment with 15-deoxy-δ- prostaglandin J₂ (15d-PGJ₂ 10 μM) or troglitazone (TGZ 30 μM) (n=6). Experiments were performed in the absence of fetal bovine serum. Data were standardised by the expression level of β-actin. (A) Expression of c-myc. (B) Expression of c-jun. (C) Expression of gadd153. **p<0.01 versus control.

Figure 8

Interaction between cell survival signalling and peroxisome proliferator activated receptor γ (PPARγ) signalling. Experiments were performed in the presence of 10% fetal bovine serum (FBS). (A) Effects of wortmannin (10 μM), an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), in combination with troglitazone (TGZ) on HT-29 cell viability in the presence of 10% FBS. Cells were treated with different concentrations of TGZ (1–100 μM) in the presence and absence of wortmannin for 24 hours (n=5–6). **p<0.01 wortmannin (−) versus wortmannin (+). (B) Effects of PD98059 (20 μM), an inhibitor of mitogen activated protein kinase, in combination with TGZ on HT-29 cell viability in the presence of 10% FBS. Cells were treated with different concentrations of TGZ (1–100 μM) in the presence and absence of PD98059 for 24 hours (n=6–8). **p<0.01 PD98059 (−) versus PD98059 (+). (C) Effects of NS-398 (20 μM), an inhibitor of cyclooxygenase 2, in combination with TGZ on HT-29 cell viability in the presence of 10% FBS. Cells were treated with different concentrations of TGZ (1–100 μM) in the presence and absence of NS-398 for 24 hours (n=6–8).