Bladder cancer is the second most common malignancy affecting the genitourinary tract. High-grade bladder cancer has an increased rate of tumour invasion, recurrence and progression. Despite recent therapeutic advances, it has a poor prognosis with a reduced 10-year survival (98 % grade 1 vs. 35 % grade 3). Extracellular nucleotides, such as ATP, have been shown to inhibit the growth of various adenocarcinomas including prostate cancer (Janssens & Boeynaems 2001). We undertook experiments to assess whether this anti-neoplastic activity extended to other urological malignancies by assessing the effect of purinergic agonists on the grade 3 human bladder cancer cell line HT-1376, a transitional cell carcinoma.

HT-1376 cells were grown in MEM supplemented with 8 % fetal bovine serum, 1 % non-essential amino acids and 1 % antibiotic solution containing penicillin and streptomycin in humidified 5 % CO₂ at 37 °C. Culture wells were seeded at 90 000 cells per well. After 24 h (day 0) cells were treated daily with ATP (10^-6 M to 10^-2 M, n = 9 for each concentration) or without ATP (control, n = 9) and incubated for a further 72 h, at which stage the number of adherent viable cells that excluded trypan blue were counted using a haemocytometer. Experiments were repeated in the presence of the P1-receptor antagonist 8-sulphophenyl theophylline (ATP 10^-4 M, 8-SPT 10^-5 M to 10^-4 M, n = 6 for each combination). The effects of the different purinergic agonists UTP, UDP, BzATP, ATPλS, 2-meSATP, 2-meSADP and α,β-meATP (all at 10^-4 M, n = 12 per agonist) on cell growth were also assessed. Results are expressed as means ± S.E.M. and statistical differences are determined using Student’s two-tail paired t test.

After 72 h the number of cells in the control group had increased 3-fold (baseline control 100 000 ± 2500 cells vs. 72 h control 296 000 ± 5 340 cells; P < 0.0001). ATP (10^-4 M) added daily not only inhibited this proliferation, but further reduced the cell number to 19 % below the baseline (72 h ATP treated wells 81 000 ± 2700 cells vs. 72 h control; P < 0.0001). This anti-neoplastic action was dose-dependent (EC₅₀ = 8.4 X 10^-5 M) and was not affected by 8-SPT indicating that ATP was acting via a P2-purinergic receptor. The rank order of agonist potency was ATPλS > ATP ▓ge│ BzATP > 2-meSATP. The P2X agonist α,β-meATP (P2X₁, P2X₃), and the P2Y agonists 2-meSADP (P2Y₁), UTP (P2Y₂, P2Y₄) and UDP (P2Y₆) had no anti-neoplastic effect.

Our results show, for the first time, that extracellular nucleotides completely inhibit the growth of urinary transitional cell carcinomas. This anti-neoplastic action is mediated by P2-purinergic receptors. The agonist profile and the rank order of agonist potency suggest this action is likely to be mediated by P2X₄, P2X₅, and/or P2Y₁₁ receptors. Exploitation of these findings may provide a potential therapeutic target for the treatment of high-grade bladder cancer.

M.S. is supported by a Royal College of Surgeons of England Research Fellowship Grant.