Long-Term Therapeutic Effects of 225Ac-DOTA-E[c(RGDfK)]2 Induced by Radiosensitization via G2/M Arrest in Pancreatic Ductal Adenocarcinoma

<b>Background</b>: Alpha radionuclide therapy has emerged as a promising novel strategy for cancer treatment; however, the therapeutic potential of ²²⁵Ac-labeled peptides in pancreatic cancer remains uninvestigated. <b>Methods</b>: In the cytotoxicity study, tumor cells were incubated with ²²⁵Ac-DOTA-RGD₂. DNA damage responses (γH2AX and 53BP1) were detected using flowcytometry or immunohistochemistry analysis. Biodistribution and therapeutic studies were carried out in BxPC-3-bearing mice. <b>Results</b>: ²²⁵Ac-DOTA-RGD₂ demonstrated potent cytotoxicity against cells expressing α_vβ₃ or α_vβ₆ integrins and induced G2/M arrest and γH2AX expression as a marker of double-stranded DNA breaks. ²²⁵Ac-DOTA-RGD₂ (20, 40, 65, or 90 kBq) showed favorable pharmacokinetics and remarkable tumor growth inhibition without severe side effects in the BxPC-3 mouse model. In vitro studies revealed that 5 and 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ swiftly induced G2/M arrest and elevated γH2AX expression. Furthermore, to clarify the mechanism of successful tumor growth inhibition for a long duration in vivo, we investigated whether short-term high radiation exposure enhances radiation sensitivity. Initially, a 4 h induction treatment with 5 and 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ enhanced both cytotoxicity and γH2AX expression with 0.5 kBq/mL of ²²⁵Ac-DOTA-RGD₂ compared to a treatment with only 0.5 kBq/mL of ²²⁵Ac-DOTA-RGD₂. Meanwhile, the γH2AX expression induced by 5 or 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ alone decreased over time. <b>Conclusions</b>: These findings highlight the potential of using ²²⁵Ac-DOTA-RGD₂ in the treatment of intractable pancreatic cancers, as its ability to induce G2/M cell cycle arrest enhances radiosensitization, resulting in notable growth inhibition.