PPARgamma agonists inhibit growth and expansion of CD133+ brain tumour stem cells

Abstract

Brain tumour stem cells (BTSCs) are a small population of cells that has self-renewal, transplantation, multidrug resistance and recurrence properties, thus remain novel therapeutic target for brain tumour. Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists induce growth arrest and apoptosis in glioblastoma cells, but their effects on BTSCs are largely unknown. In this study, we generated gliospheres with more than 50% CD133+ BTSC by culturing U87MG and T98G human glioblastoma cells with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In vitro treatment with PPARgamma agonist, 15-Deoxy-Delta(12,14)-Prostaglandin J(2) (15d-PGJ2) or all-trans retinoic acid resulted in a reversible inhibition of gliosphere formation in culture. Peroxisome proliferator-activated receptor gamma agonists inhibited the proliferation and expansion of glioma and gliosphere cells in a dose-dependent manner. Peroxisome proliferator-activated receptor gamma agonists also induced cell cycle arrest and apoptosis in association with the inhibition of EGF/bFGF signalling through Tyk2-Stat3 pathway and expression of PPARgamma in gliosphere cells. These findings demonstrate that PPARgamma agonists regulate growth and expansion of BTSCs and extend their use to target BTSCs in the treatment of brain tumour.

Figure 1

Inhibition of gliosphere formation by PPARγ agonists. The T98G and U87MG human glioblastoma cells were cultured in DMEM medium with 10% FBS (A1 and E1) or NBM with B27 and 10 ng ml⁻¹ EGF+bFGF in the absence (B1 and F1) or presence of 1.0 μM 15d-PGJ2 (C1 and G1) or ATRA (D1 and H1) and photographed ( × 200) after 10 days. The cells were then cultured in fresh DMEM medium with 10% FBS (A2, B2 and E2, F2) or NBM with B27 and 10 ng ml⁻¹ EGF+bFGF in the absence of 15d-PGJ2 (C2 and G2) or ATRA (D2 and H2) and photographed ( × 200) after 10 days. The T98G and U87MG cells were also cultured in NBM with B27 and EGF+bFGF in the absence or presence of Ciglitazone, 15d-PGJ2 and ATRA, the number of gliospheres counted after 10 days and the mean±s.e.m. of triplicates presented in the histogram. The P-values are shown as (^*P<0.05), (^**P<0.01) and (^***P<0.001) and the figure is a representative for three independent experiments.

Figure 2

Inhibition of CD133+ BTSC expansion by PPARγ agonists. The U87MG and T98G-sphere cells were cultured in NBM+B27 with EGF+FGF in the absence (A) or presence of 1 μM ciglitazone (B), 15d-PGJ2 (C) or ATRA (D). After 5 days, the cells were stained with anti-CD133-PE or isotype-matched control antibody and analysed by flow cytometer. The percentage of CD133+ cells and mean fluorescence intensity in brakets are shown.

Figure 3

Inhibition of BTSC proliferation by PPARγ agonists. The U87MG and T98G cells (A, B) and the gliosphere cells (C, D) were cultured in NBM+B27 with different concentrations of EGF+FGF. The U87MG and T98G cells were cultured in NBM with B2 and 10 ng ml⁻¹ bFGF+EGF in the presence of ciglitazone (E, F), 15d-PGJ2 (I, J) or ATRA (M, N) in 96-well tissue culture plates in triplicate. U87MG- and T98G-gliosphere cells were also cultured in NBM+B27 with 10 ng ml⁻¹ EGF+FGF in the presence of ciglitazone (G, H), 15d-PGJ2 (K, L) or ATRA (O, P). After 48 h, ³H thymidine (0.5 μCi per well) was added, and the amount of ³H thymidine uptake was counted on a Perkin Elmer Microbetaplate liquid scintillation counter as a measure of proliferation. The values are mean of triplicate (±s.e.m.) with P-values shown as (^*P<0.05), (^**P<0.01) and (^***P<0.001). The figure is representative of three independent experiments.

Figure 4

Induction of cell cycle arrest and apoptosis by PPARγ agonists in BTSCs. U87MG and T98G-sphere cells (2 × 10⁶ cells) were cultured in NBM with B27 and EGF+FGF in the presence of 1 μM ciglitazone, 15d-PGJ2 or ATRA. The cells were harvested after 48 h and stained with propidium iodide (A), Annexin V-FITC (B) or dUTP-FITC (C) and analysed by flow cytometry. Genomic DNA was extracted and analysed by agarose gel electrophoresis to visualise DNA fragmentation (D). The figure is representative of three independent experiments.

Figure 5

Inhibition of EGF/FGF signalling and induction of PPARγ expression by PPARγ agonists in BTSCs. T98G-sphere cells (2 × 10⁶ cells) were cultured in NBM with B27 and EGF+FGF in the absence or presence of ciglitazone, 15d-PGJ2 or ATRA. The cells were harvested after 15 min and the whole cell lysates were analysed by SDS–PAGE and western blot using phosphospecific Tyk2 or Stat3 antibodies and visualised by ECL detection system (A). The cells were harvested after 36 h and total RNA was analysed for the expression of PPARγ by real-time RT–PCR using GAPDH as internal control (B). The values are mean of triplicate (±s.e.m.) and P-values shown as (^*P<0.05) in the figure. The cells were harvested after 48 h and total protein was analysed for the expression of PPARγ analysed by SDS–PAGE and western blot using anti-PPARγ antibodies and visualised by ECL detection system (C). The figure is representative of three independent experiments.