Radiation cystitis results from radiation therapy for pelvic tumors and accounts for up to 7% of emergency urology admissions.  It can be classified into acute and chronic stages where the former is characterized by urothelial layer disruption and inflammation.  Current therapies are limited, invasive and often fail to demonstrate optimal efficacy.  Radiation damage is mediated by reactive oxygen and nitrogen species.  Our findings suggest that the principal site damage in the bladder is urothelial mitochondria and mitochondrial-targeted free radical scavengers are radioprotective when instilled into the bladder for delivery to urothelial cells.  Since radiotherapy is usually delivered over a treatment course by means of multiple fractions, frequent catheterizations can increase the risks for urinary tract infections and irritation.  This is why in this study we examined the duration of the protective effect of XJB-5-131 against urothelial damage during five days of fractionated irradiation.  A similar compound lacking the hemigramicidin S tag targeting it to mitochondria, JP4-039, was also tested.

To determine the duration of the protective effect of XJB-5-131 and JP4-039 against urothelial damage, we used human immortalized uroepithelial cells (UROtsa).  Cultured cells at 10-15 passage were plated in 48 well plates at 150 cells/well and subjected to fractionated irradiation (2 Gy/day/5 days).  Cells were treated with 1 µM XJB-5-131, 1 µM JP4-039 or vehicle (0.01% DMSO) once prior to the first irradiation dose or every day. Following each irradiation, cells at the center of each well (≈0.3 cm2 area) were imaged using a tissue culture microscope (Fig 1) and counted by Olympus CellSens software (green areas on right panels of Fig 1). Results from 8 wells were averaged for each group, experiment repeated 5 times.

Our data demonstrate a dose-dependent cell death over the course of irradiation treatment to only about 40% of cells treated with vehicle surviving at the end of 5 days (Fig 2).  Non-irradiated cells grew to ~300% of the original number.  Both groups of XJB-5-131 treated irradiated cells (those treated once or every day) grew to ~200% cells over first 5 days.  In both groups treated with JP4-039 the growth slowed down after day two of irradiation treatment and cell death prevailed thereafter.

XJB-5-131 was protective when administered once at a start of a 5 days week of fractionated irradiation, with no benefit if given daily, thus limiting the likelihood of urinary tract infections and irritations due to repeated catheterizations.  It is important for the free radical scavenger to be targeted to the mitochondria since non-targeted compound did not offer similar protection.

Mitochondrial-targeted free radical scavenger, XJB-5-131, is protective against radiation cystitis when given on the first day of weekly fractionated radiation therapy.