Phenylbutyrate induces apoptosis and lipid accumulations via a peroxisome proliferator-activated receptor gamma-dependent pathway

Abstract

The effects of the selective peroxisome proliferator activated receptor-gamma (PPAR-gamma) inhibitor GW9662 on phenylbutyrate (PB)-induced NMR-detectable lipid metabolites was investigated on DU145 prostate cancer cells. DU145 cells were perfused with 10 mM PB in the presence or absence of 1 microM of GW9662 and the results monitored by (31)P and diffusion-weighted (1)H NMR spectroscopy. GW9662 completely reversed PB-induced NMR-visible lipid and total choline accumulation in (1)H spectra and glycerophosphocholine and beta-NTP in (31)P spectra. In addition, pre-incubation with GW9662 significantly reduced PB-induced caspase-3 activation, reversed the G(1) block as measured by flow cytometry, and otherwise had little effect on cell survival as measured by MTT assay. These results suggest that the NMR visible lipid accumulation and apoptosis induced by PB treatment occurs through a mechanism that is mediated by PPAR-gamma.

Figure 1

Diffusion-weighted ¹H NMR spectra (left traces) and ³¹P NMR spectra (right traces) of perfused DU145 prostate cancer cells treated with A) control at 16 h B) 1 µM GW9662 for 16 h; C) 10 mM PB for 16 h; D) 1 µM GW9662 and 10 mM PB for 16 h. The 0 ppm peak in ¹H spectra results from the microcarrier beads used as a growth support for the cells. Additional peak assignments are as shown.

Figure 2

Time dependent changes in the area of the A) methylene resonance at 1.3 ppm; B) the tCho resonance at 3.2 ppm; C) the GPC resonance at 0.5 ppm and D) the β-NTP resonance at −18 ppm of perfused DU145 cells as measured by ¹H and ³¹P NMR. Changes in resonance areas were scaled to the intracellular water resonance at the analogous time point and are reported as changes relative to time zero. Open circles indicate cells treated with 1 µM GW9662 and 10 mM PB, filled circles indicate control cells treated with GW9662 alone, open squares indicate cells treated with 10 mM PB alone, filled squares indicate untreated control cells.

Figure 3

Caspase-3 activation (a) and MTT dye reduction (b) in DU145 cells. Caspase-3 activation was significantly higher in DU145 cells treated with PB alone relative to untreated controls (*; p< 0.05), GW9662 only treated cells, and PB + GW9662 treated cells. In the MTT assay, no significant difference was observed in the cytotoxicity of cells treated with PB alone or PB + 1 µM GW9662.

Figure 4

Flow cytometric DNA histograms showing cell cycle distribution in DU145 cells treated with PB in the presence or absence of GW9662

Figure 5

Cell cycle distribution of DU145 cells treated with PB and 0, 5 and 10 µM of GW9662. Significance is indicated by †: significantly different from control, p< 0.05; ††: significantly different from control; p< 0.01; *: significantly different from PB-treated, p< 0.05; **: significantly different from PB-treated, p< 0.01; §: significantly different from PB-treated at equivalent GW9662 concentration.