Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in human transitional bladder cancer and its role in inducing cell death

Abstract

The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

Figure 1

In situ hybridization showing distribution of PPARγ and RXRα mRNA in human bladder cancer tissue. (a) Dark-field illumination of uninvolved tissue of human bladder; white grains depict hybridization over urinary transitional epithelium; original magnification, x50. (b) Dark-field illumination of in situ hybridization for PPARγ over involved human bladder cancer tissue; white grains indicate hybridization over bladder epithelial cell layer and submucosai infiltrating malignant transitional cells (arrow); original magnification, x50. (c) Human bladder cancer tissue (bright field); black grains indicate hybridization over submucosai infiltrating malignant bladder cancer cells; original magnification, x400. (d) Dark-field illumination of uninvolved tissue of human bladder; white grains depict hybridization over urinary epithelial cells; original magnification, x10. (e) Dark-field illumination of in situ hybridization for PPARγ over involved human bladder cancer tissue; white dots indicate hybridization over bladder epithelial cell layer and submucosal infiltrating malignant transitional cells; original magnification, x20. (f) Human bladder cancer tissue (bright field); black grains indicate hybridization over infiltrating malignant bladder cancer cells; original magnification, x400.

Figure 2

Upper panel, RNase protection showing RXRα mRNA expression (335-bp protected band) in four human bladder cancer cell lines (RT-4, T24, 5637, and HT-1376) and one transformed human ureter epithelial cell line (SV-HUC-1). Middle and lower panels, immunoblot and nuclease protection showing PPARγ protein expression in five transitional cell lines and PPARγ mRNA in five human transitional cell lines, kidney, and adipose tissue. Lower panel, a 350-bp protected band was detected in all cell lines. Middle panel, PPARγ protein was also detected by immunoblot with a doublet at ∼55 kDa.

Figure 3

Troglitazone inhibits ³H-thymidine incorporation and decreases viability of T24 human bladder cancer cells. Upper panel, concentration-dependent effects of troglitazone on ³H-thymidine incorporation in T24 cells. ³H-thymidine incorporation was measured as described in Materials and Methods section. Results are expressed as percentage inhibition compared to untreated cells. Error bars represent standard errors of mean (SEM) (n=16; *P<0.05, **P<0.01). Data are from a single experiment and are representative of four independent experiments. Lower panel, concentration-dependent effects of troglitazone on viability of T24 cells were determined by the MTT assay. Values represent the mean±SEM of 12 wells from a single experiment representative of three independent experiments (n=12; *P<0.05, **P<0.01).

Figure 4

(A) Representative photomicrographs showing morphology of T24 bladder cancer cells before treatment (left panel) and after a 24-hour treatment with 15 µM troglitazone (right panel). (B) Induction of apoptosis in T24 bladder cancer cells treated with 15 µM troglitazone for 1 day. The TUNEL assay was performed, labeling the apoptosis-specific DNA strand breaks with green fluorescent label as described in Materials and Methods section. Left panel, T24 cells treated with DMSO. Right panel, T24 cells treated with troglitazone (15 µM) for 24 hours.

Figure 5

The RXRα ligand, 9-cis -RA, sensitizes T24 bladder cancer cells to troglitazone or 15dPGJ₂-induced cell death. Upper panel, quiescent T24 cells were treated with troglitazone (0.1, 1, 5, 10, 15, and 20 µM) in the presence or absence of 9-cis -RA LG100268 (5 µM) for 24 hours. MTT assay was performed to measure cellular viability. Values represent the percentage of inhibition of control culture (treated with vehicle alone) from five independent experiments. Error bars are standard errors of mean (SEM) (n=20; *P<0.05, **P<0.01). Lower panel, concentration-dependent effects of 15dPQJ₂ on T24 cell viability. Study was performed in the presence or absence of 9-cis -RA (5 µM). Values represent the percentage of control culture (treated with vehicle alone) values from three independent experiments. Error bars are standard errors of mean (SEM) (n=12; *P<0.05, **P<0.01).

Figure 6

Nuclease protection assay showing effects of troglitazone on mRNA expression of cell cycle regulatory proteins in T24 bladder cancer cells. Quiescent T24 cells were treated with troglitazone (10 µM) for 1, 4, 8, and 24 hours. Total RNA was extracted and analyzed as described under Materials and Methods section. Upper panels, nuclease protection for cyclin-dependent kinase inhibitor, p16^INK4 (338 bp), p21^WAF1/CIP1 (420 bp), and cyclin D1 (391 bp). Lower panels, RNA loading was assessed by simultaneous use of a β-actin (125 bp) riboprobe.

Figure 7

PPARγ is functionally active in T24 bladder cancer cells. Cells were transfected with PPRE3-Luc with control vector or PPARγ expression vector and treated with 10 µM troglitazone or control buffer for 14 hours. Results are mean±SEM of n=7, representative of three independent experiments. Control values are normalized to 1.0. Asterisks indicate significant difference from the control (*P<0.05, **P<0.01).

Figure 8

Panel A, effect of troglitazone on expression of A-FABP in bladder cancer cells. Cells were treated with troglitazone (10 µM) for 7 days and mRNA expression was determined by RNase protection. Lane 1, probes for β-actin and A-FABP, vehicle alone, Lane 2, troglitazone (10 µM). Total RNA (5 µg) was used for detection and the β-actin was the control for the amount of loaded RNA. Panel B, in situ hybridization showing distribution of A-FABP mRNA (white grains) in normal uroepithelial cell layer (original magnification, x40, top panel) and in submucosal infiltrating bladder cancer cells (original magnification, x400, lower panel).