Cyclophosphamide Induces an Early Wave of Acrolein-Independent Apoptosis in the Urothelium

Abstract

Purpose: Hemorrhagic cystitis (HC or bladder inflammation) affects a significant number of patients undergoing cyclophosphamide (CP) chemotherapy despite treatment with 2-mercaptoethane sulfonate (Mesna) to inactivate the metabolite acrolein. While the mechanism is unknown, there is clearly acrolein-independent damage to the urothelium. In this study we have explored the induction of apoptosis in the urothelium as a marker of damage and the mechanism underlying the acrolein-independent apoptosis.

Materials and methods: Apoptosis in urothelium (caspase-3/7 activity and Poly (ADP-ribosyl) polymerase (PARP) cleavage) was measured following CP administration (80 mg/kg). Sodium 2-mercaptoethane sulfonate (Mesna) was used to mask acrolein's effect. An IL-1β receptor antagonist and a cell-permeable caspase-1 inhibitor were used to assess the involvement of IL-1β and caspase-1, respectively.

Results: Two waves of apoptosis were detected following CP administration, one peaking at 2 h and a second at 48 h. The first wave was independent of acrolein. Caspase-1 was also active at 2 h and activation of caspase-3/7 was blocked by a caspase-1 inhibitor but not an IL-1β receptor antagonist suggesting the direct activation of caspase-3/7 by caspase-1 without the need for IL-1β as an intermediate.

Conclusions: Our results indicate that CP initiates an early, acrolein-independent wave of apoptosis that results from direct cleavage of caspase-3/7 by caspase-1.

Keywords: Apoptosis; Bladder; Cyclophosphamide; Cystitis; Urothelium.

Figure 1

a. Activity of caspase-3/7 in rat urothelium over time following injection of 80 mg/kg CP. Data points are the mean ±SEM. Asterisks indicate values significantly different from 0 h (p<0.0006 at 2h, <0.02 at 4 h, <0.0015 at 16 h, <0.014 at 24 h, <0.0001 at 48 h and < 0.007 at 72h). n = 3-6 replicates. b. Flow cytometry histograms showing the cell population staining positive (under the M1 gate) for cleaved PARP in rat urothelium 0 and 2 h following 80 mg/kg CP. c. Quantitative analysis of b. Data points are the mean ± SEM. Asterisks indicate significant differences from 0 h control. n = 5; p<0.05.

Figure 2

Ability of Mesna to block caspase-3/7 activity in urothelium 2 and 24 h after CP treatment. a. Experimental protocol for treatment of rats with Mesna, CP and vehicle. b. Caspase-3/7 activity in the samples indicated in a. Data points are the mean ± SEM. * indicate values significantly different from the mean value at 0 h. (p<0.05). n = 4 for 0 h and both 2 h time points. n=6 for 24 h – mesna and 3 for 24 h + mesna. # indicates the 24h + mesna group is significantly different from the 24 h – mesna group (p<0.05). All other comparisons were not significant.

Figure 3

a. Caspase-1 activity in rat urothelium 2 h after injection of 80 mg/kg CP or saline control. Data points are the mean ± SEM. Asterisks indicate values significantly different from the mean value at 0 h, n = 6, p<0.05. b. (upper) diagram of the protocol for treatment with YVAD-AOM , CP and DMSO. b. (lower) caspase-3/7 activity in the various treatment groups. Data points are the mean ± SEM. Asterisks indicate values significantly different from DMSO control; n=6, p<0.05. No other differences between groups were significant. c. (upper) diagram of the protocol for treatment with Anakinra (Ana), CP and saline. c. (lower) caspase-3/7 activity in the various treatment groups. Data points are the mean ± SEM. Asterisks indicate values significantly different from saline control; n=5-6, p<0.05. No other differences between groups were significant.