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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Keywords: 225Ac; DNA damage; RGD peptide; integrin; pancreatic cancer; radionuclide therapy.

Figure 1

In vitro cytotoxicity. (a) Cytotoxicity of ²²⁵Ac-DOTA-RGD₂ in human pancreatic tumor cell lines. (b) Comparison of cytotoxicity between ²²⁵Ac-DOTA-RGD₂ and ²²⁵AcDOTA in BxPC-3. All assays were performed in triplicate. Data are presented as mean ± standard deviation.

Figure 2

Induction of γH2AX and 53BP1 foci formation in response to increasing doses of ²²⁵Ac-DOTA-RGD₂ at 24 h. (a) Representative images of γH2AX and 53BP1 foci obtained by immunofluorescence microscopy in BxPC-3 cells. Scale bar, 20 μm. (b) The number of γH2AX and 53BP1 foci per cell. Induction of γH2AX and 53BP1 foci in response to increasing doses of ²²⁵Ac-DOTA-RGD₂ was monitored at 24 h. The number of γH2AX and 53BP1 foci per cell was counted, and 50–100 cells were analyzed. All assays were performed in triplicate. Data are presented as mean ± standard deviation and analyzed using a one-way analysis of variance with Dunn’s multiple-comparisons test (* p < 0.05, ** p < 0.01).

Figure 3

Flow cytometric analysis of BxPC-3 after incubation with ²²⁵Ac-DOTA-RGD₂. (a) Representative fluorescence-activated cell sorting plots for γH2AX. The y-axis indicates γH2AX staining, and the x-axis is the DNA content. (b) Percentage of cells with γH2AX staining. All assays were performed in triplicate. (c) Percentage of cell cycle distribution (G1, S, and G2/M). All assays were performed in triplicate. Data are presented as the mean ± standard deviation (* p < 0.05, ** p < 0.01, and **** p < 0.0001).

Figure 4

Biodistribution of ²²⁵Ac-DOTA-RGD₂ in BxPC-3-bearing mice. (a) Pharmacokinetics of ²²⁵Ac-DOTA-RGD₂. Data are expressed as % ID/g for organs and blood and as % ID for carcass, urine, and feces. Data are shown as the mean ± standard deviation (n = 3–4). (b) Alpha camera imaging of intratumoral distribution and corresponding hematoxylin and eosin images. The scale bars indicate 100 μm. ID, injected dose.

Figure 5

Therapeutic efficacy of ²²⁵Ac-DOTA-RGD₂ in BxPC-3-bearing mice. (a) Individual tumor responses. Each solid color line represents a tumor from a single mouse. (b) Relative tumor growth of the mice groups treated with a single dose of ²²⁵Ac-DOTA-RGD₂ compared to the control group (untreated). Data are shown as the mean ± standard deviation. (c) Kaplan–Meier survival curves of the mice treated with ²²⁵Ac-DOTA-RGD₂. Log-rank (Mantel–Cox) test; p = 0.0192, hazard ratio [HR] 2.415, 95% CI 0.7639–7.636 (control vs. 20 kBq); p = 0.0014, HR 3.342, 95% CI 0.9631–11.60 (control vs. 40 kBq); p = 0.0002, HR 3.774, 95% CI 1.042–13.67 (control vs. 65 kBq); p = 0.0009, HR 3.786, 95% CI 1.062–13.49 (control vs. 90 kBq). (d) Change in body weight after administration of ²²⁵Ac-DOTA-RGD₂. Data are shown as the mean ± standard deviation.

Figure 6

Cytotoxicity, cell cycle, and γH2AX expression by low-dose ²²⁵Ac-DOTA-RGD₂ after 4 h of treatment with high-dose (5 or 10 kBq/mL) of ²²⁵Ac-DOTA-RGD₂ in BxPC-3 and PANC-1 cells. (a) Cell viability. The white, grey, and blue columns indicate the pretreatment with 0, 5, and 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂, respectively. (b) Percentage of cell cycle distribution. (c) Time course of γH2AX expression. The significance of γH2AX expression at each time point was compared to 0, 5, or 10 kBq/mL as the control in each graph. Data represent the mean ± standard deviation (n = 2–4). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.