Low-Dose Difluoromethylornithine and Polyamine Levels in Human Prostate Tissue

Difluoromethylornithine is a noncompetitive inhibitor of ornithine decarboxylase, which catalyzes the ratelimiting step in polyamine synthesis. Induction of ornithine decarboxylase activity has been closely associated with the action of tumor promoters (1,2), growth factors (3,4), and mitogenic hormones (5–7). Difluoromethylornithine inhibits the development of spontaneous and carcinogen-induced mammalian tumors in vivo, including carcinomas of the breast, colon, epidermis, and bladder (8–11). At low difluoromethylornithine doses of 0.5 g/m or less given by mouth daily, polyamine suppression has been demonstrated in skin and colonic tissues (12–14), with limited and reversible toxicity (13,14). The prostate gland is of particular interest with respect to difluoromethylornithine because adult mammalian prostatic tissue has been reported to have high activities of ornithine decarboxylase (5) and elevated concentrations of polyamines (15). These become even more elevated in mammalian prostatic carcinoma (15,16). We studied the effects of 2 weeks of oral, daily, lowdose (0.5 g/m) difluoromethylornithine on human peripheral zone prostatic ornithine decarboxylase activities and polyamine levels in patients undergoing open prostatic surgery. Under an institutionally approved protocol and after written informed consent, men undergoing radical prostatectomy with biopsy-proven diagnoses of prostatic cancer (n 4 24) or cystoprostatectomy for bladder cancer without prostatic cancer (n 4 1), whose serum creatinine levels were 1.5 mg/100 mL or less and who had no important major organ system or hearing deficits, were randomly assigned in a 3 : 2 ratio to receive oral difluoromethylornithine given at 0.5 g/m once daily (n 4 15) or no treatment (n 4 10) for 2 weeks. One difluoromethylornithine-treated case patient was not evaluable. Plasma testosterone, serum prostate-specific antigen (PSA), and prostatic acid phosphatase levels were measured and volar forearm punch skin biopsies were performed as previously described (17) before the drug was started and 12–14 hours after the final dose was given. At surgery, 12– 14 hours after the last ingested dose of difluoromethylornithine, after the prostate gland was removed the prostate gland was immediately sampled using a True-cut biopsy needle ex vivo. Tissues that contained suspicious prostatic nodules were sampled with a minimum of one core taken from the peripheral zone from each side of the prostate (12 patients). In 12 patients, three similar cores were taken from each side of the prostate. Each core, roughly 2.0 mm in diameter and 18 mm long, was divided longitudinally into thirds. The middle section was placed in 10% formalin for subsequent histologic inspection, and the end sections were analyzed for ornithine decarboxylase activities and polyamine concentrations. Tissue specimens were homogenized in ice-cold buffer (50 nM Tris–HCl [pH 7.5], 0.1 mM EDTA, and 0.1 mM pyridoxal phosphate). Ornithine decarboxylase activity in the clear supernatant and tissue polyamines were measured by previously reported methods (12,13). Plasma testosterone, serum PSA, and serum prostatic acid phosphatase levels were measured by standard laboratory techniques. The primary analysis concerned examining differences in ornithine decarboxylase activity and polyamine levels in prostate tissue between the difluoromethylornithine-treated and control groups. The values assigned to each subject were the average of the samples or the geometric mean when logarithmic transformations were used. Differences in the mean ornithine decarboxylase activity in prostate tissue were examined by a two-sample t test. Because the variance of polyamine measurements appeared to increase directly with their concentrations, polyamine levels were compared after logarithmic (base 10) transformations were taken. The logtransformed data were then analyzed with two-sample t tests. The difluoromethylornithine-treated subjects (n 4 14) completed 2 weeks of treatment without any reported toxic effects. Comparisons of pretreatment groups and post-treatment groups with respect to pretreatment clinical stage and post-treatment pathologic stage, tumor grades (Gleason scores), and levels of PSA, prostatic acid phosphatase, and testosterone showed no evidence of a treatment effect. Mean putrescine concentrations in prostate tissue were statistically significantly lower in the difluoromethylornithine-treated group (log [nmol/mg DNA]: difluoromethylornithine-treated group 4 1.43; control group 4 1.95; two-sided P 4 .03). Prostatic ornithine decarboxylase, spermidine, and spermine concentrations were comparable in the two groups. Skin ornithine decarboxylase levels tended to be lower after treatment in the difluoromethylornithine-treated group (not statistically significant). Although the numbers of prostatic tissue cores ranged from two to six in individual subjects and a majority of tissue cores did not show cancer despite specific attempts to sample palpable cancer nodules, no associations of difluoromethylorni th ine t rea tment wi th these parameters were observed. Both the polyamine biosynthetic and the interconversion pathways regulate polyamine levels (18,19). Difluoromethylornithine inhibits ornithine decarboxylase activity and reduces putrescine and occasionally spermidine levels. Thus, it may not be surprising that only putrescine levels were found to be depressed in this study. It is uncertain whether a longer duration of administration of difluoromethylornithine would