Antineoplastic effects of rosiglitazone and PPARgamma transactivation in neuroblastoma cells

Abstract

Neuroblastoma (NB) is the most common extracranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. In the present study, we evaluated the role of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone (RGZ) in two NB cell lines (SK-N-AS and SH-SY5Y), which express PPARgamma. Rosiglitazone decreased cell proliferation and viability to a greater extent in SK-N-AS than in SH-SY5Y. Furthermore, 20 microM RGZ significantly inhibited cell adhesion, invasiveness and apoptosis in SK-N-AS, but not in SH-SY5Y. Because of the different response of SK-N-AS and SH-SY5Y cells to RGZ, the function of PPARgamma as a transcriptional activator was assessed. Noticeably, transient transcription experiments with a PPARgamma responsive element showed that RGZ induced a three-fold increase of the reporter activity in SK-N-AS, whereas no effect was observed in SH-SY5Y. The different PPARgamma activity may be likely due to the markedly lower amount of phopshorylated (i.e. inactive) protein observed in SK-N-AS. To our knowledge, this is the first demonstration that the differential response of NB cells to RGZ may be related to differences in PPARgamma transactivation. This finding indicates that PPARgamma activity may be useful to select those patients, for whom PPARgamma agonists may have a beneficial therapeutic effect.

Figure 1

(A and B) Effect of a 24- or a 48-h treatment with RGZ on ³[H]thymidine incorporation in SK-N-AS (A) and SH-SY5Y (B) cells. ^*P<0.05 vs control untreated cells (C). (C and D) Effect of a 24- or a 48-h treatment with RGZ on SK-N-AS (C) and SH-SY5Y (D) cell proliferation. ^*P<0.05 vs control untreated cells (C).

Figure 2

(A and B) Role of RGZ on SK-N-AS (A) and SH-SY5Y (B) cell viability, as assessed by MTS assay (see Materials and Methods). ^*P<0.05 vs control untreated cells (C). (C and D) Effect of different concentrations and time of exposure to RGZ (24 or 48 h) on SK-N-AS (C) and SH-SY5Y (D) cell adhesion, as assessed by Bengal Rose assay (see Materials and Methods). ^*P<0.05 vs control untreated cells (C).

Figure 3

Role of the PPARγ antagonist BADGE (indicated as B) (20 μM) in counteracting the effect on ³[H]thymidine incorporation (A) and on cell viability (B) induced by 20 μM RGZ in SK-N-AS cells. ^*P<0.05 vs control untreated cells (C).

Figure 4

Role of RGZ on SK-N-AS cell invasiveness. (A) Untreated control cells. (B) Cells treated with 20 μM RGZ for 24 h. The invasive cells on the lower surface of the membrane were stained with crystal violet (see Materials and Methods). (C) Spectrophotometric analysis on extracts from stained cells eluted from the membrane invasion chamber. ^*P<0.05 vs control untreated cells (C).

Figure 5

Amount of MMP-9 mRNA (A) and protein (B) and of TIMP-1 mRNA (C) in SK-N-AS cells in the absence or in the presence of different concentrations of RGZ. ^*P<0.05 vs control (indicated as C).

Figure 6

Immunocytochemistry for the detection of cleaved caspase 3-positive cells. (A and C) Control untreated cells (SK-N-AS and SH-SY5Y, respectively). (B and D) RGZ-treated cells (SK-N-AS and SH-SY5Y, respectively). Magnification × 40.

Figure 7

PPARγ transcriptional activity in control untreated NB cells (C), in cells treated with RGZ (20 μM) and in cells transfected with PPARγ in the absence or in the presence of RGZ. L/C: peroxisome proliferator response element-n7₃-tk-luciferase reporter activity, normalised for CAT activity. ^*P<0.05 vs C. ^**P<0.05 vs PPARγ transfected cells in the absence of RGZ.

Figure 8

(A) Western blot analysis of the amount of PPARγ in SK-N-AS and SH-SY5Y. PANC-1 and HPAC cells (human pancreatic adenocarcinomas) were used as the positive and negative control, respectively. (B) Coomassie R-stained gel, showing equal protein loading of the same samples indicated in A. (C) Detection of total (anti-PPARγ antibody) and phosphorylated (anti-P-Ser antibody) PPARγ, after PPARγ immunoprecipitation.