Evaluation of a Rhenium(I) Complex and Its Pyridostatin-Containing Chelator as Radiosensitizers for Chemoradiotherapy

Abstract

The use of radiosensitizers is a beneficial approach in cancer radiotherapy treatment. However, the enhancement of radiation effects on cancer cells by radiosensitizers involves several different mechanisms, reflecting the chemical nature of the radiosensitizer. G-quadruplex (G4) DNA ligands have emerged in recent years as a potential new class of radiosensitizers binding to specific DNA sequences. Recently, we have shown that the Re(I) tricarbonyl complex PDF-Pz-Re and its pyrazolyl-diamine chelator PDF-Pz, carrying a N-methylated pyridostatin (PDF) derivative, act as G4 binders of various G4-forming DNA and RNA sequences. As described in this contribution, these features prompted us to evaluate PDF-Pz-Re and PDF-Pz as radiosensitizers of prostate cancer PC3 cells submitted to concomitant treatment with Co-60 radiation. The compound RHPS4 was also tested, as this G4 ligand was previously shown to exhibit strong radiosensitizing properties in other cancer cell lines. The assessment of the resulting radiobiological effects, namely through clonogenic cell survival, DNA damage, and ROS production assays, showed that PDF-Pz-Re and PDF-Pz were able to radiosensitize PC3 cells despite being less active than RHPS4. Our results corroborate that G4 DNA ligands are a class of compounds with potential interest as radiosensitizers, deserving further studies to optimize their radiosensitization activity and elucidate the mechanisms of action.

Keywords: Co-60 irradiation; G4-DNA binders; Re(I) complexes; pyridostatin derivatives; radiosensitizers.

Figure 1

Structures of the G4-DNA ligands evaluated in this work as radiosensitizers of PC3 PCa cells.

Figure 2

Cellular viability of PC3 cells after 48 h of incubation with the compounds PDS, PDF, PDF-Pz, and PDF-Pz-Re [21] and with RHPS4 at different concentrations (0.5–100 µM). Data are expressed as the percentage of cellular viability (mean ± SEM).

Figure 3

Inhibitory effect of PDS (20 µM), PDF (5 µM), PDF-Pz (30 µM), PDF-Pz-Re (30 µM), and RHPS4 (2.5 µM), in combination with Co-60 irradiation, on the proliferation of PC3 cells. After incubation with the compounds for 48 h, PC3 cells were exposed to 0.5, 1, 2, 4, and 6 Gy of γ-radiation (dose rate of 1 Gy/min). IR-only control experiments were also performed for each tested radiation dose. The results were calculated from independent biological replicates (n = 3) and are given as the mean ± SEM.

Figure 4

DNA damage induced in PC3 cells treated with Co-60 irradiation at 2 and 4 Gy doses after previous incubation with 30 μM of PDS-Pz and PDS-Pz-Re, for 48 h at 37 °C. Control cells were incubated with medium only. (A) Quantification of γ-H2AX foci per nucleus (mean ± SEM, 2 independent experiments), expressed as the average number of foci. Statistical differences compared to the control are denoted as not significant (ns) and * p < 0.05, ** p < 0.01 (B) Representative fluorescence images of cells exposed to the different radiation doses in the presence or not of the tested compounds. (C) Distribution of γ-H2AX foci across samples, presented as the relative frequency of nuclei with a certain number of foci.

Figure 5

ROS production in PC3 cells treated with PDF-Pz or PDF-Pz-Re (30 µM). Cells were incubated with the compounds and then irradiated at 2 and 4 Gy. Similar experiments were run for cells treated uniquely with IR, at the same radiation doses. Cells treated with H₂O₂ (10 μM) were used as a positive control. ROS production was measured 1 h post-irradiation. Non-irradiated cells (0 Gy) under IR-only conditions served as the control group (not significant (ns); **** p ≤ 0.0001). The results were calculated from independent biological replicates (n = 3) and are given as the mean ± SEM.