Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity

Abstract

l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

Figure 1

Effects of pretreatment with 1 nM l-deprenyl on the radiation response of human normal and malignant bladder explants. The area of the outgrowth was measured on day 14 postirradiation and the treated outgrowth area is expressed as a percentage of the control (mean±s.e.m. for five patients per group). Absolute values for the normal control were 460±120 mm² and for the tumour 246±60 mm². ^*P<0.05, ^**P<0.01.

Figure 2

Effects of varying concentrations of l-deprenyl and varying radiation doses on the % of HaCaT or HaCaT-ras cell colonies surviving treatment. Cells were plated in medium containing 1 nM l-deprenyl and irradiated 6 h later. Absolute control plating efficiency values were 34.5±2.7 for HaCaT cells and 56.7±3.7 for HaCaT-ras cells.

Figure 3

Clonogenic survival for HaCaT and HaCaT-ras cells treated with cisplatin (μg ml⁻¹) for 1 h. Cells were plated in medium containing 1 nM l-deprenyl and exposed to cisplatin 16 h later. Absolute control plating efficiencies were 23.6±1.3 for HaCaT cells and 52.2±2.1 for HaCaT-ras cells.

Figure 4

(A) Expression of Bcl-2 protein in normal human uroepithelial explants, treated as indicated with 1 nM l-deprenyl or clorgyline. The values for the tumour samples were all 100%, and are not shown. (B) Apoptosis postirradiation in primary urothelial cultures treated with l-deprenyl. Data are mean percent counts of apoptotic cells in 10 randomly selected fields per culture.

Figure 5

Expression of apoptosis (A) and bcl-2 (B) in HaCaT cells with and without l-deprenyl treatment prior to irradiation. Cultures were all seeded with 400 cells and were fixed at 48 h postirradiation to 0, 0.5 or 5 Gy Co-60 gamma rays. Apoptosis and Bcl-2 expression were scored in the same microcolonies. In all, 20 microcolonies were scored for each culture. Five replicate cultures were counted. Data were pooled due to low frequency of the end points. The figures show the relative frequency of apoptotic and Bcl-2-positive cells in the microcolonies. Open symbols, solid lines: − deprenyl; Closed symbols, broken lines: + deprenyl. Cells per microcolony: 1 (□, ▪); 2 (○, •); 3 (◊, ⧫); 4 (▵, ▴).