Redox-Active Cerium Fluoride Nanoparticles Mitigate of Double-Stranded DNA Breakage and Modulate Gene Expression Due to Radiation Exposure In Vitro

1. Introduction

Ionizing radiation, which is commonly used in cancer therapy, is electromagnetic radiation or photons that ionize the molecules in the cell [1]. Ionized molecules are highly reactive species undergoing a rapid cascade of chemical transformations leading to the breaking of chemical bonds [2]. As a result, the DNA structure, the main target of ionizing radiation, is disrupted, and highly reactive products are formed from water molecules contained in the cell, such as reactive oxygen species (ROS) and free radicals [3]. Consequently, exposure to ionizing radiation directly and indirectly leads to a damage to the basic structures of the cell and its death. However, ionizing radiation also triggers a series of signaling cascades that promote cell survival and activate cellular processes such as DNA repair, cell cycle arrest and autophagy, which leads to radioresistance and the development of tumor cell adaptability [4].

Selective increase of radiosensitivity of tumor cells is one of the important tasks of the development of a new modern treatment methods of solid tumors radiation therapy [5]. There are a few approaches to increase the radiation sensitivity of cancer cells, starting with increasing the level of tumor tissue oxygenation, ending with the use of kinase inhibitors (for example, PI3K-like kinases) or cell cycle checkpoints [6]. The most promising agents for these purposes is the use of nanoradiosensitizers based on high Z elements, which can effectively accumulate in tumor cells and locally enhance the radiation dose, thereby increasing the damaging effect of ionizing radiation [7]. Some of them have already been approved by the FDA or are at various stages of clinical trials [8,9]. Thus, the use of nanoscale particles with a good biosafety profile and the ability to effectively absorb ionizing radiation is a promising strategy in the development of new effective radiosensitizers.

Fluoride nanoparticles have their unique physicochemical properties, which determines such interest in the study. In particular, lanthanide fluorides (LnF₃, Ln = lanthanides) have very low solubility (about 10^–5–10^–6 mol/L) [10] and, as a consequence, low toxicity [11]. For example, previous studies in vivo on planarians confirm that CeF₃ nanoparticles have low toxicity at concentrations of 1 μM - 10 mM [12]. Cerium fluoride is one of the best scintillators for use in biomedicine [13]. Scintillators are compounds that are capable of emitting photons when they absorb ionizing radiation of various types, including X rays. CeF₃ NPs is an effective scintillator, since when irradiated with X-rays, it emits UV light due to fluorescence at a wavelength of 325 nm. In the nanoscale state, cerium ions are able to transfer excitation energy to ions in its doped crystal lattice, for example, rare-earth ions of other metals (for example, europium or terbium) located in the crystal lattice [14]. In [15], the cytotoxicity of Ce³⁺, Tb³⁺: NaYF₄ spherical particles against Capan-1 cells with a size of 16.7 nm was studied in the concentration range of 0.5 pM - 40 nM using the MTT assay. After 48 h of incubation, a dose-dependent linear decrease in viability was detected from ~100% at 0.5 pM to ~30% at 40 nM. According to the general opinion, cerium-containing nanoparticles with a predominant content of cerium in the trivalent state on the surface provide their SOD-mimetic activity [16]. Trivalent cerium ions determine the redox-dependent anti-apoptotic effect. For example, PEGylated CeO₂ nanoparticles with high Ce³⁺ content protect cells from ROS-induced cell death in vitro and reduce the rate of ischemic cell death in vivo [17]. Soh et al. synthesized 2 nm cerium zirconium oxide (Ce_0.7Zr_0.3O₂) nanoparticles with a high concentration of Ce³⁺ ions, which demonstrated anti-inflammatory activity in two representative models of sepsis [18]. In addition, both undoped and terbium-doped CeF₃ nanoparticles have been shown to provide effective cell protection against vesicular stomatitis virus [11]. CeF₃ NPs were shown to prevent oxidative discoloration of indigo carmine dye induced by exposure to hydrogen peroxide, where a dose-dependent protective effect was detected. Luminescent nanocrystals containing lanthanide Ln³⁺ ions are currently of interest to researchers because of their unique characteristics, including a long luminescence lifetime, high resistance to photochemical decomposition and photobleaching, as well as high chemical and physical stability [19,20,21]. In addition, these nanocrystals have the ability to form stable colloids [22]. CeF₃:Tb@LaF₃ based nanomaterials are promising biomedical agents due to their high colloidal stability and resistance to photobleaching [23,24]. Coating of CeF₃:Tb³⁺ NPs with LaF₃ shells improves the emission intensity by more than 25% compared to conventional CeF₃:Tb³⁺ nanoparticles. Energy losses from the surface luminescence centers were significantly reduced due to the LaF₃ shell, which served as a barrier for energy transfer to the upper surface of the shell, and therefore the lifetime and luminescence intensity of these nanoparticles significantly increased [25]. The luminescent and magnetic properties of lanthanide nanoparticles may be of fundamental importance in the development of multifunctional nanomaterials, allowing them to be used in MRI and other luminescence methods [26,27]. CeF₃ NPs can be considered as an effective platform for development of promising redox-active drugs that can find their application in the therapy and diagnosis of oncological diseases.

In this work, redox-active CeF₃ NPs were used to analyze their impact to cell proliferation, mitochondrial membrane potential and DNA repair process upon X-ray irradiation. The effect CeF₃ NPs on the rate of DSBs in normal and cancer cells was analyzed and the possible molecular mechanisms of this effect were discussed. The significant improvement of the DSBs DNA repair rate by CeF₃ NPs in normal cells exposed to ionizing radiation shows new opportunities in development of advanced radiation-mediated cancer therapies.

2. Materials and Methods

2.1. Synthesis and Characterization of CeF₃ NPs

CeF₃ NPs were synthesized by precipitation in alcoholic media [11]. UV–vis absorption spectra of the colloid solution was recorded using standard quartz cells for liquid samples on a UV5 Nano spectrophotometer (Mettler Toledo, USA). Transmission electron microscopy and selected area electron diffraction (SAED) analysis was performed using a Leo 912 AB Omega electron microscope operating at 100 kV acceleration voltage. Nanoparticle size measurements by dynamic light scattering and zeta-potential were carried out on a BeNano Zeta particle size analyzer (BetterSize, China). Powder X-ray diffraction analysis (XRD) of CeF₃ NPs sol (dried at 40°C) was performed using a Bruker (Billerica, MA, USA) D8 Advance diffractometer (CuKα radiation), in the angle range of 20-90°2 Θ, with a step of 0.02°2Θ and a signal acquisition time of 0.4 s per step. Full-profile analysis of XRD patterns was performed using TOPAS v.4.2 software (Billerica, MA, USA) and diffraction maxima were fitted to Voigt pseudo-functions.

2.2. Cell Cultures

The experiments were performed using a culture of human mesenchymal stem cells (hMSc) derived from the third molar bud, extracted from a healthy 12-year-old patient for orthodontic indications, and MCF-7 human breast cancer cell line, obtained from Theranostics and Nuclear Medicine Laboratory cryostorage (ITEB RAS, Pushchino, Russia). The in vitro experiments were performed in agreement with good clinical practice and the ethical principles of the current edition of the Declaration of Helsinki, after the permission of the Ethics Committee of Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences (Pushchino, Moscow region, Russian Federation), protocol No. 35 from 5 March 2022. Postnatal human dental pulp stem cells were extracted from the third molar tooth of a human donor (of an 12-year-old donor). The tooth was removed according to the orthodontic indications of the dental clinic «Dr. MUN» (Moscow, Russia), in accordance with the ethics committee, after consent was signed by the patient’s parents. The cells were cultivated in a DMEM/F12 (1:1) medium containing 10% of fetal bovine serum (FBS), 50 μg/mL of penicillin, 50 μg/mL of streptomycin and 1% of l-glutamine, at 37 °C in a humid atmosphere, comprising 95% air and 5% CO₂. The cells were seeded at a density of 20,000–35,000/cm². The medium was replaced by a medium containing CeF₃ NPs in different concentrations (10^-3 –10^-7 M) 6 h after cell attachment. In the control experiments, the cells were cultured without CeF₃ NPs.

2.3. X-ray Irradiation

X-ray irradiation of the cell cultures was performed using an X-ray therapeutic machine RTM-15 (Mosrentgen, Russia) in a dose of 1 and 5 Gy for CeF₃ NPs solution, 15 Gy for cell cultures, 1.5 Gy for DSBs analysis at a dose rate of 1 Gy/min, 200 kV voltage, 37.5 cm focal length and a 20 mA current.

2.4. Assessment of CeF₃ NPs Antioxidant Activity

To evaluate the antioxidant activity of CeF₃ NPs, we determined the concentration of hydrogen peroxide after X-ray irradiation of CeF₃ NPs by enhanced chemiluminescence using a luminol – 4-iodophenol – peroxidase system [28]. We used TRIS buffer to maintain constant pH 7.2. The irradiation dose was 5 Gy (1 Gy per min). A liquid scintillation counter Beta-1 (MedApparatura, Ukraine), operating in the mode for counting single photons (with one photomultiplier and the coincidence scheme disengaged), was used as a highly sensitive chemiluminometer. The high sensitivity of this method allows registering H₂O₂ at a concentration of < 1 nM. The H₂O₂ content was determined using calibration dependencies of chemiluminescence on H₂O₂ concentration in the solution. The concentration of H₂O₂ used for the calibration was determined spectrophotometrically at 240 nm using a molar absorption coefficient of 43.6 M⁻¹ × cm⁻¹. The hydroxyl radical under X-ray irradiation was measured using coumarin-3-carboxylic acid [29]. The fluorescence intensity of hydroxylated coumarin-3-carboxylic acid (ex 395/em 450 nm) was measured on a Cary Eclipse spectrofluorimeter (Agilent, Australia) at room temperature.

2.5. Cell Proliferation Analysis

The cell growth analysis after incubation with CeF₃ NPs and X-ray irradiation was estimated by counting the number of the cells stained with Hoechst 33342 using fluorescent imager Zoe^® (BioRAd, USA). At least four measurements were made for each CeF₃ NPs (10^‒3-10^‒7 M). The data are presented as growth curves (mean with SD).

2.6. Analysis of DNA Double-Strand Breaks by γ-H2AX Foci

Cells were seeded in 35 mm Petri dishes with a central hole (Ibidi, Germany). After X ray irradiation the cells were washed twice with phosphate-buffered saline (PBS, 3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, pH 7.4). Then the cells were fixed with 4 % paraformaldehyde in PBS for 15 min, permeabilized with 0.1 % Triton X-100 in PBS for 5 min, washed extensively with PBS and blocked in 5% BSA in PBS (Gibco, Invitrogen, Karlsruhe, Germany) for 30 min at room temperature (RT). The primary antibodies, monoclonal mouse γH2AX (ab195188 Recombinant, Anti-gamma H2A.X (phospho S139), (Alexa Fluor® 488), Abcam) were used for immunostaining. Image acquisition of the DSBs was performed using an inverted fluorescent microscopy Zeiss Axiovert Observer 200 (Carl Zeiss Microscopy, Jena, Germany). Analysis of γH2AX stained cells was performed with an optional manual correction in individual cells. For each experimental group, at least 200 cells were analyzed. Image J software was used to quantify the detected γH2AX foci.

2.7. RT-PCR (Genes Expression Analysis)

Reverse transcription was performed with a Evrogen kit (Russia, Moscow), using an oligodT primer according to the manufacturer’s protocol. For PCR reaction, a mixture was used with SYBR Green dye (Evrogen, Russia). To conduct a PCR-RT analysis, a BioRad CFX-96 amplifier Real-Time PCR System was used. The expression of 96 genes responsible for key cell processes under oxidative stress was thus determined (Table S1). For PCR profiling, the genes to be analyzed were selected from the database http://www.sabiosciences.com/. The level of gene transcription was normalized by the levels of expression of housekeeping genes β-actin, rplp0 (ribosomal protein, large, P0) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The gene-specific primers were prepared using Genesis software. Each measurement was made twice (internal repetition) and averaged for two independent samples. A sample without a reverse transcription stage was used as the control. The expression data obtained were analyzed using online services http://www.sabiosciences.com/, mayday-2.14 software (Center for Bioinformatics, Tübingen, Germany) and Genesis software.

2.8. Statistical Analysis

The experiments were conducted in 3–4 repetitions, with three independent repetitions for each CeF₃ NPs concentration. Experimental results were compared with untreated control. Statistical analysis was performed using the methods of variation statistics (ANOVA, Student's t-test.). The mean values and the standard deviation (SD) of the mean were determined. The obtained data were processed using the GraphPad 8.0 Software.

3. Results and Discussion

The synthetic procedures resulted in stable aqueous sol of highly crystalline CeF₃ NPs. Selected area electron diffraction patterns of the samples (Figure 1a) correspond to the P6_3/mcm space groups for hexagonal CeF₃ structure. High crystallinity of CeF₃ NPs synthesized using soft chemistry routes is typical for this Ce(III)-containing material [11]. According to XRD results, the resulting sol contains as a dispersed phase pure hexagonal cerium fluoride (sp. gr. P63/mcm) with a crystallite size of 24 ± 2 nm(Figure 1b). ζ-potential of CeF₃ NPs sol equals +41 mV, corroborating its high stability (Data not presented). The hydrodynamic radii of CeF₃ NPs in the sol diluted with distilled water was 40±2 nm (Figure 1c), indicating the low degree of the particle agglomeration. According to TEM data, the size of CeF₃ NPs was 15–25 nm. The nanoparticles possess notable faceting, which indicates high degree of crystallinity. Highly crystalline CeF₃ NPs is expected to contain only trivalent cerium [30,31]. The main UV absorption peak of CeF NPs is located at 250 nm (Figure 1d). According to literature data, UV absorption spectra of Ce^{3 +} ions have a maximum at 253.6 nm with a molar extinction coefficient of 685 M^{− 1} cm^{− 1} [32]. Data presented in Figure 1e shows that CeF₃ NPs are capable of preventing the formation of hydrogen peroxide in water upon X-ray exposure due to their catalase-like properties. CeF₃ NPs at a concentration of 10^-7 M do not lead to a decrease in the level of hydrogen peroxide formed after irradiation of the solution. An increase in the concentration of CeF₃ NPs to 10^-6 M provides a pronounced antioxidant effect, which is expressed in a significant (up to 30%) decrease in the concentration of hydrogen peroxide. CeF₃ NPs at a concentration of 10^-5 M reduced the level of hydrogen peroxide after irradiation to zero values. A possible explanation for such CeF₃ NPs catalase-like activity can be explained by the number of binding sites for hydrogen peroxide, which directly correlates with the nanoparticle size. Catalytic decomposition of hydrogen peroxide is a heterogeneous process that occurs at the interface and depends on the specific surface area. CeF₃ NPs have a size of 15-25 nm, which corresponds to ≈ 10% of surface cerium atoms [33,34]. Thus, the specific surface area of a CeF₃ NPs occupies a smaller part of its entire surface. CeF₃ NPs reduce the concentration of hydroxyl radical (Figure 1f), which is formed during irradiation only in high concentrations (10^-5 M).

CeF₃ NPs selectively modulate the proliferative activity of normal and tumor cells (Figure 2). Preincubation of hMSCs with CeF₃ NPs leads to an increase in their proliferative activity (Figure 2a,b). Irradiation of hMSCs leads to inhibition of proliferative activity (reduction to 40% relative to non-irradiated control), however, cell pretreatment with CeF₃ NPs ensures the preservation of positive dynamics of proliferative activity at concentrations (10^-3-10^-4 M), which confirms the pronounced radioprotective effect. At the same time, the proliferative activity of tumor cells significantly decreases in the presence of CeF₃ NPs on the 5th day of coincubation, and X ray irradiation (15 Gy dose) enhances this effect (Figure 2с,d). Thus, CeF₃ NPs, on the one hand, stimulate the proliferative activity of normal cells, and, on the other, suppress the proliferative activity of tumor cells and have a radiosensitizing effect on them. Presumably, the above-described biological activity of CeF₃ NPs can be realized by their effect on the modulation of various intracellular molecular cascades that differ in normal and tumor cells.

The effect of ionizing irradiation on eukaryotic cells leads to disruption of the electron transport chain system in mitochondria, which leads to delayed radiation-induced MMP collapse and increased generation of reactive oxygen species (ROS) [35,36]. Increased generation of ROS in mitochondria and a decrease in ATP synthesis in irradiated cells can be observed for a long time [37]. Thus, damaged mitochondria become inducers of oxidative stress over a long period of time. Using the cationic dye tetramethylrhodamine (TMRE), which accumulates in mitochondria in a voltage-dependent manner, we analyzed the mitochondrial membrane potential (MMP) of cell cultures 72 hours after irradiation (Figure 3). It was revealed that irradiation of hMSCs at a dose of 15 Gy does not lead to a significant decrease in MMP (Figure 3а). Pretreatment of cells with CeF₃ NPs also does not affect the MMP level after irradiation. It is worth noting that after irradiation we observe morphological changes, in particular, the enlargement of mitochondria, which confirms the development of the senescence process. Pretreatment of cancer cells with CeF₃ NPs leads to a slight decrease in the level of MMP, but no statistically significant decrease is observed. Irradiation of tumor cells leads to hyperpolarization of mitochondria (increase in MMP), and their pretreatment with CeF₃ NPs nanoparticles only enhances this effect in a dose-dependent manner (Figure 3b). Thus, the combined effect of CeF₃ NPs and X-ray irradiation have exactly the opposite effect on the mitochondrial membrane potential of tumor cells.

Ionizing radiation generally leads to diverse DNA damages, including single-strand and double-strand breaks [3]. Double-strand breaks (DSBs) make up a small part of such damage, while it is their number that is decisive for the further fate of the cell including the cell cycle arrest, DNA repair processes activation or apoptosis triggering [38]. Using γ-H2AX histone staining with FITC-labeling antibodies, we evaluated the dynamics of the DSBs repair in normal and cancer cells after irradiation. Figure 4 shows that the number of double-strand breaks significantly reduced in the presence of CeF₃ NPs in both normal (hMSCs) and cancer (MCF-7) cells 1 hour after irradiation. Then, after 4 hours of incubation, the dynamics of the repair of DSBs become different for normal and cancer cells. For normal cells, the protective effect of CeF₃ NPs is observed for all CeF₃ NPs concentrations, which is expressed in a decrease in the number of DSBs (Figure 4 a, c). Meanwhile, for MCF-7cancer cells, there is a significant increase of DSBs in comparison with the irradiated control (Figure 4 b, d).

The effect of CeF₃ NPs on the expression pattern in both cancer (MCF-7) and normal (hMSc) cells revealed downregulation of most genes (49 out of 89) (Figure 5). For example, genes coding some members of the glutathione peroxidase family (GPX1, GPX2, GPX5) were significantly down-regulated. Irradiation of MCF-7 cells in the presence of CeF₃ NPs revealed upregulation in 13 genes, as well as suppressed expression of 17 studied genes. PCA analysis revealed a clear effect of radiation on the pattern of gene expression, and its pronounced modulation in the presence of CeF₃ NPs. It should be noted that the separate action of irradiation or CeF₃ NPs similarly influenced the expression patterns for both types of cell cultures, while the combined action of CeF₃ NPs and X-ray irradiation was fundamentally different for cancer and normal cells. This difference can be associated with the effect of CeF₃ NPs on the signaling pathways of the cell, in particular, on NF-κB. NF-κB is a transcription factor that participates in the regulation of various physiological processes, including response to oxidative stress, inflammation and apoptosis [39]. The activation mechanisms of NF-κB pathway are associated with inhibition of IκB subunit. Earlier, it has been shown that CeO₂ NPs decrease intracellular oxidative stress and suppress phosphorylation of IκBα and the translocation of P65 subunits of NF-κB into the cell nuclei, which provide cell protection under cigarette smoke exposure [40].

Thus, we hypotized that CeF₃ NPs are able to modulate the activity of NF-κB both in the direction of activation and in the direction of suppression. NF-κB signaling pathway is also responsible for the repair of DSBs after X ray exposure [41]. In cancer cell (MCF-7 cells), inhibition of the IKK-NFκB pathway by a specific inhibitor of IKKβ was found to significantly reduce the repair rate of irradiation-induced DSBs and sensitized MCF-7 cells to clonogenic cell death. Recent studies show that CeO₂ NPs are able to influence key cell signaling pathways, regulating the processes of proliferation, apoptosis, differentiation, protein synthesis and cell cycle progression [42]. Thus, the study of cerium-containing nanoparticles effect on cell signaling pathways could be one of the ways to increase the sensitization of cancer cells to ionizing radiation, increasing the efficiency of radiation therapy of oncological diseases.

5. Conclusions

CeF₃ NPs significantly reduce the content of hydrogen peroxide and hydroxyl radicals after X-ray exposure. CeF₃ NPs in normal cells act as a proliferation stimulator and a radioprotector reducing the number of DSBs, maintaining high viability, proliferative activity and mitochondrial membrane potential maintaining viability, proliferative activity and mitochondrial membrane potential close to control values after irradiation. Meanwhile, in cancer cells CeF₃ NPs act as proliferation suppressor and a radiosensitizer, which slows down the rate of DSBs repair 4 h after irradiation. CeF₃ NPs reduce the proliferative activity of cancer cells, lead to radiation-induced hyperpolarization of mitochondria. The molecular mechanisms of the selective action of CeF₃ NPs are most likely associated with the modulation of intracellular signaling pathways through the regulation of the redox status of the cell which we confirmed by RT-PCR analysis. The observed selective enhancement of DSBs repair process of cerium-containing nanoparticles can be used to increase the sensitization of cancer cells in a patient during radiation therapy and to protect healthy tissues in the immediate vicinity of the tumor.