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.
Highlights
The goal of radiation therapy (RT) is to maximize the probability of tumour control whilst minimizing damage to surrounding normal tissue
Radiation therapy (RT) for bladder, prostate and cervical pelvic malignancies is associated with radiation-induced bladder toxicity (RIBT) which typically manifests as lower urinary tract symptoms [1,2,3,4] due to unavoidable dose delivered to neighbouring normal tissues
Radiation bystander effects have been described in normal bladder explants correlated with aberrant urothelial outgrowth [6,7,8,9] which may be a protective response to urothelial loss that occurs during RT [10], associated with irritative radiation cystitis [11, 12]
Summary
The goal of radiation therapy (RT) is to maximize the probability of tumour control whilst minimizing damage to surrounding normal tissue. RT for bladder, prostate and cervical pelvic malignancies is associated with radiation-induced bladder toxicity (RIBT) which typically manifests as lower urinary tract symptoms [1,2,3,4] due to unavoidable dose delivered to neighbouring normal tissues. Many cells repair DNA damage through homologous recombination or nonhomologous end joining to maintain genome integrity while others, including tumour cells lack effective repair mechanisms and undergo cell death. Radiation bystander effects have been described in normal bladder explants correlated with aberrant urothelial outgrowth [6,7,8,9] which may be a protective response to urothelial loss that occurs during RT [10], associated with irritative radiation cystitis [11, 12]. In the context of urothelial cancer, studies using the bladder transitional cancer cell line, EJ138, have demonstrated decreased clonogenic cell survival after transfer of medium from irradiated cells, an effect which saturated at 2Gy, consistent with a bystander response [13,14,15]
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