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 4 MBq/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 P2Y6-selective 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.
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