Extracellular ATP Release Triggered by ¹³¹I-Trastuzumab Mitigates Radiation-Induced Reduction in Cell Viability through the P2Y₆ Receptor in SKOV3 Cells

Intracellular ATP is released outside cells by various stimuli and is involved in cytoprotection by activating purinergic receptors. However, it remains unclear whether targeted radionuclide therapy induces extracellular ATP release. Here, we prepared ¹³¹I-labeled trastuzumab (¹³¹I-trastuzumab) and examined extracellular ATP release and its roles in ¹³¹I-trastuzumab’s growth inhibitory effects. ¹³¹I-trastuzumab was prepared by labeling with the chloramine-T method. The binding of ¹³¹I-trastuzumab to cells was investigated using the human epidermal growth factor receptor 2 (HER2)-positive cells (SKOV3) and the HER2-negative cell (MCF7). Extracellular ATP was determined by measuring chemiluminescence using a luciferin-luciferase reagent. The growth inhibitory effects of ¹³¹I-trastuzumab were investigated by colony formation assay. ¹³¹I-trastuzumab bound exclusively to SKOV3 cells. Treatment with ¹³¹I-trastuzumab at 4MBq/mL and higher concentrations significantly increased extracellular ATP levels, whereas non-radioactive trastuzumab didn’t. This suggested that ATP release was specifically induced by radiation derived from ¹³¹I. The growth inhibitory effects of ¹³¹I-trastuzumab were significantly enhanced by pretreatment with apyrase (ecto-ATPase) or MRS2578 (a P2Y₆-selective antagonist), whereas they were significantly reduced by treatment with a P2Y₆selective agonist. In conclusion, ¹³¹I-trastuzumab induced extracellular ATP release, and the released ATP was shown to be involved in mitigating radiation-induced reduction in cell viability through P2Y₆ receptor.