Purinergic impact on urothelial cell proliferation

P Aronsson, M Andersson, G Tobin. University of Gothenburg, Department of Pharmacology, 40530, Sweden

Introduction: In the current study we investigated the effects of adenosine 5\'triphosphate (ATP), an anti-proliferative agent in the prostatic gland (1), and adenosine on the rate of proliferation on an immortalized human urothelial cell-line, designated UROtsa.

Aim: The aim of the investigation was to establish whether or not ATP and adenosine have receptor-mediated effects on the proliferation of urothelial cells, compared to the basal rate of proliferation. If so, can the specific purinergic receptor subtypes responsible be characterized?

Method and Materials: Cells of the UROtsa cell-line were cultivated in Dulbecco´s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37C in a 5/95% CO2/O2 atmosphere. Confluent cell cultures were trypsinized and 40.000 cells were transferred to each well of a 96-well plate. The cells were allowed to attach for 2 hours after which drugs were added and the plate was incubated over night.

The following day the MTT Cell Proliferation Assay was used to quantitatively measure the drug-induced changes in proliferation rate, equivalent to the amount of absorbance at 570 nm. Untreated cells and medium not containing cells were used as control and blank respectively.

Drugs used in this study were the P2 agonist ATP, P2X desensitizer alpha,beta-meATP (abMeATP), P2 purinergic antagonists PPADS and suramin, P1 agonist adenosine, P1A1 antagonist DPCPX, P1A2B antagonist PSB1115 and A2 agonist CV1808. Statistics were calculated using 1-way ANOVA with Bonferroni’s Post test. Values are expressed as mean±SEM.

Results and Discussion: Both ATP and adenosine significantly and concentration-dependently inhibited cell proliferation (at 10-4 M: -12±2; p<0.01, n=10 and -14±2% p<0.0001, n=9; respectively). The ATP-induced anti-proliferative effect (at 10-4 M) was antagonized by suramin (from 95±6 to 120±3%, at 10-6 and 10-4 M suramin, respectively, p<0.05, n=4) and PPADS (from 88±2 to 102±2%, at 10-6 and 10-4 M PPADS, respectively, p<0.0001, n=9). The P2X desensitizer abMeATP however seemed to lack effect (from 86±6 to 87±6%, at 10-6 and 10-3 M abMeATP, respectively n.s, n=5).

Regarding the P1 receptors, the antagonists DPCPX and PSB1115 had no or only minute effects (93±7 and 94±12% at 1.5x10-6 and 10-4 M respectively, n.s, n=4), on the adenosine-evoked anti-proliferatory effects (10-4M). However, the P1A2 agonist CV1808 (10-8 to 10-5M; 95±1% at 10-8 M p<0.05, n=7) had a small antiproliferative effect.

Thus, in the bladder urothelium ATP exerts anti-proliferatory effects via P2Y purinoceptor mediated signals, while this preliminary data may indicate that the adenosine effect involves P1A2 purinoceptors. However, further experiments need to be conducted in order to fully characterize the specific subtype involvement.

References:

(1) Shabbir M, Thompson C, Jarmulowiczc M, Mikhailidis D, Burnstock G. Effect of extracellular ATP on the growth of hormone-refractory prostate cancer in vivo. BJU Int. 2008;102:108–112.