Dual effects of radiation bystander signaling in urothelial cancer: purinergic-activation of apoptosis attenuates survival of urothelial cancer and normal urothelial cells

Abstract

Radiation therapy (RT) delivers tumour kill, directly and often via bystander mechanisms. Bladder toxicity is a dose limiting constraint in pelvic RT, manifested as radiation cystitis and urinary symptoms. We aimed to investigate the impact of radiation-induced bystander signaling on normal/cancer urothelial cells. Human urothelial cancer cells T24, HT1376 and normal urothelial cells HUC, SV-HUC were used. Cells were irradiated and studied directly, or conditioned medium from irradiated cells (CM) was transferred to naïve, cells. T24 or SV-HUC cells in the shielded half of irradiated flasks had increased numbers of DNA damage foci vs non-irradiated cells. A physical barrier blocked this response, indicating release of transmitters from irradiated cells. Clonogenic survival of shielded T24 or SV-HUC was also reduced; a physical barrier prevented this phenomenon. CM-transfer increased pro-apoptotic caspase-3 activity, increased cleaved caspase-3 and cleaved PARP expression and reduced survival protein XIAP expression. This effect was mimicked by ATP. ATP or CM evoked suramin-sensitive Ca²⁺-signals. Irradiation increased [ATP] in CM from T24. The CM-inhibitory effect on T24 clonogenic survival was blocked by apyrase, or mimicked by ATP. We conclude that radiation-induced bystander signaling enhances urothelial cancer cell killing via activation of purinergic pro-apoptotic pathways. This benefit is accompanied by normal urothelial damage indicating RT bladder toxicity is also bystander-mediated.