p53-stabilizing agent CP-31398 prevents growth and invasion of urothelial cancer of the bladder in transgenic UPII-SV40T mice

Abstract

The high prevalence of bladder cancer and its recurrence make it an important target for chemoprevention. About half of invasive urothelial tumors have mutations in p53. We determined the chemopreventive efficacy of a p53-stabilizing agent, CP-31398, in a transgenic UPII-SV40T mouse model of bladder transitional cell carcinoma (TCC) that strongly resembles human TCC. After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm of CP-31398 for 34 weeks. Progression of bladder cancer growth was monitored by magnetic resonance imaging. At 40 weeks of age, all mice were killed; urinary bladders were collected to determine weights, tumor incidence, and histopathology. There was a significant increase in bladder weights of transgenic versus wild-type mice (male: 140.2 mg vs 27.3 mg, P < .0001; female: 34.2 mg vs 14.8 mg, P < .0001). A significant decrease in the bladder tumor weights (by 68.6-80.2%, P < .0001 in males and by 36.9-55.3%, P < .0001 in females) was observed in CP-31398-treated mice. Invasive papillary TCC incidence was 100% in transgenic mice fed control diet. Both male and female mice exposed to CP-31398 showed inhibition of invasive TCC. CP-31398 (300 ppm) completely blocked invasion in female mice. Molecular analysis of the bladder tumors showed an increase in apoptosis markers (p53, p21, Bax, and Annexin V) with a decrease in vascular endothelial growth factor in transgenic mice fed CP-31398. These results suggest that p53-modulating agents can serve as potential chemopreventive agents for bladder TCC.

Figure 1

(A) Structure of p53-stabilizing agent, CP-31398. (B) Experimental design for chemopreventive efficacy evaluation of CP-31398 in UPII-SV40T transgenic mice. At 6 weeks of age, groups (30 per group UPII-SV40T or 12 per group wild type) of mice were fed experimental diets containing 0, 150, or 300 ppm of CP-31398 continuously for 34 weeks and bladders from each mouse were evaluated histopathologically and also for expression of various markers as described in the text.

Figure 2

Upper panels: (A) Urinary bladders of the UPII-SV40T mice increase in both size and weight compared with the wild-type mice. (B) Terminal body weights of the transgenic mice fed control or experimental diets. (C) Male and (D) female bladder weights (means ± standard error) at the termination of the experiment. The differences between the wild-type and UPII-SV40T animals fed control diet were analyzed by unpaired t test with Welch correction. Lower panels: Typical histologic characterization of urothelium showing (E) normal wild-type or (F) low-grade noninvasive, (G) high-grade noninvasive, and (H) high-grade invasive TCC in UPII-SV40T mice upon feeding with control AIN-76A diet. No evidence of TCC was seen in the bladders of wild-type mice.

Figure 3

(A) MRI images of the urinary bladders from wild-type and UPII-SV40T transgenic mice. Transgenic mice were given either control diet or diet containing CP-31398 for 36 weeks. Morphologic and contrast-enhanced imaging (Gd-DTPA administered through an i.v. tail vein catheter) MRI was performed on these mice at 40 weeks of age. Wild-type urinary bladders do not show any tumors. Untreated UPII-SV40T mice have large, dense tumor inside the bladder, while the treated transgenic mice have significantly reduced and barely visible tumor. (B and C) Effect of CP-31398 on bladder tumor weights in male (B) and female (C) mice, respectively, at the termination of the experiment. CP-31398 treatment led to a significant reduction of tumor weight in both groups. (D and E) CP-31398 treatment had an inhibitory effect on the incidence of invasive TCC in male (D) and female (E) mice, respectively.

Figure 4

Effect of CP-31398 on expression of p53, Bax, and VEGF markers. Quantitative real-time PCR analysis was done on bladder tumors of male mice as described in Materials and Methods section. (A, C, E) Relative expression of p53, Bax, and VEGF mRNA, respectively, in UPII-SV40T urothelium compared with wild type. (B, D, F) CP-31398 treatment led to an increase in p53 and Bax, while VEGF mRNA levels were inhibited.

Figure 5

Effect of CP-31398 on p21 (A–D) and Annexin V (E–H) expression in bladder tumors. Immunohistochemical analysis was performed with paraffin-embedded and microsectioned bladder tissues from male mice as described in Materials and Methods section. A significantly increased expression of p21 (A–D) and Annexin V (E–H) was seen in the treatment groups.