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1. CeO2-x and CeO2-x nanoparticles

Nanotechnology has become a promising ally for research and treatment of diseases. In addition to a wide range of established applications in multiple areas of research (1), cerium has captured the interest of researchers due to its redox properties. Cerium, a rare earth metal, can exist in both +3 and +4 states. Thus, cerium oxide (CeO2-x) can occur in two different forms: CeO2 and Ce2O3 in the bulk state, due to the coexistence of the element cerium (Ce) in two different oxidation states: Ce3+ [(Xe) 4f1] and Ce4+ [(Xe)]. The CeO2-Ce2O3 phase transition depends on the oxygen pressure and system temperature, as well as the reduction transition (2,3). Among the compounds with Ce4+, the CeO2 phase presents the most stable structure: A face-centred cubic crystalline network (FCC) of the fluorite type (Fm3m) (4,5). Each tetravalent cerium cation (Ce4+) coordinates with eight oxygen anions (O-2) (Fig. 1A), further providing greater stability compared with the hexagonal structure of Ce2O3 (6). The CeO2 structure could present intrinsic and extrinsic defects in the atom arrangement. Intrinsic defects are related to the thermal disorder of the material or the result of atmosphere surrounding the material, the redox process (7). At the nanoscale, however, the formation of non-stoichiometric oxides is observed in the redox process in CeO2-x, which creates pure CeO2-x nanoparticle structures where 0 <x <0.5(8). In this process, electrons are transferred from the oxygen anion (O2-) to the cerium cation (Ce4+), which generates an oxygen vacancy from the reduction of Ce4+ to Ce3+ (Fig. 1B; Eq. 1).

Figure 1 Schematic figure of (A) unit cell of cerium oxide with a face-centered cubic crystalline network structure of the fluorite type and (B) formation of non-stoichiometric oxide during the redox process. Ce4+ is represented by brown balls, O2- by blue balls, Ce3+ by red balls and the vacancies, by the empty circles.

(Eq. 1: ø represents the empty position due to the atom displacement in the structure after electron transfer).

An oxygen vacancy is a structural disarrangement caused by the increase or decrease of oxygen concentration inside the particle and cerium ion radius. It is important to note that the concentration of oxygen defects increase with the reduction in particle size, which confers better redox properties of CeO2-x nanoparticles compared with their oxide (9,10). In chemical catalysis, vacancy is defined as the ability of an oxide to store and release oxygen. The description of oxygen vacancies in transition oxides and rare earth oxides is an unexplored challenge for modern calculations of electronic structure (11). Due to the increase in the surface-to-volume ratio, CeO2-x nanoparticles have higher concentrations of Ce3+, compared with CeO2-x particles. Thus, there is greater mobility of oxygen in the structure for the rapid generation of surface vacancies. This property provides easy switching between Ce3+ and Ce4+ oxidation states, generating numerous active points for redox reactions to occur on the surface of nanoparticles. Due to their oxygen buffering capacity, CeO2-x nanoparticles can also self-regenerate to the initial state of Ce4+ with no side reactions (12).

Nanoparticle structural defects-vacancies

Vacancies are structural defects in a particle, which can be formed through electronic relocation in the structure of the material, generating redox hotspots. Vacancies are not restricted to the oxide surface only. For CeO2-x, studies demonstrate that the electrons resulting from the formation of oxygen vacancies on the surface and subsurface in the crystallographic plane (111; Miller index) may not go to the atoms directly linked to the electron donator atom, but to more distant atoms instead (11,13). Due to the increase in the surface area, CeO2-x nanoparticles have more Ce3+ ions on their surface and their vacancies can occur by a quantum ionization/displacement process of cerium 4f electrons, as proposed by Skorodumova et al (14). Electrons in the f subshell create redox hotspots by the reduction of Ce4+ to Ce3+ (11). In addition, the migration of electrons from 2p orbital oxygen states in the valence to the conduction band is only possible if the energy between them (band gap) is relatively small (14,15). In CeO2-x nanoparticles, vacancies are dynamic and can change spontaneously or in response to physical parameters, such as temperature, partial pressure of oxygen, doping with other ions and application of an electric field or surface stress (16,17). Another phenomenon responsible for causing vacancies is entropic stabilization, which will appear on surfaces with numerous empty spaces. Beyond particle size, some other factors may influence the redox activity of the nanoparticles, such as suspension medium and formation of agglomerates. Reducible oxide surfaces, such as CeO2-x nanoparticles, are highly disordered at the nanoscale, causing a greater formation of empty spaces, facilitating the formation of vacancies, and presenting even greater redox activity (18). Due to their high redox activity, CeO2-x nanoparticles have started to gain attention in the biomedical field, particularly with regard to diseases in which a redox imbalance is observed. It is important to note that a redox imbalance can be found in a wide range of diseases, including cancer (19). Therefore, some studies have addressed the oxidative stress-associated cytotoxic effects of CeO2-x nanoparticles in biological models, particularly focusing on the modulation of the apoptotic pathway, the most studied regulated cell death modality (Tables I and II). This is relevant, considering the increasing body of evidence characterizing genetically-regulated distinct mechanisms of cell death (20). Due to the wide diversity of cell death subroutines, the present study focuses only on the effects of nanoparticles on apoptotic cell death.

Table I Effects of cerium oxide nanoparticles in non-cancer disease models.

Table I

Effects of cerium oxide nanoparticles in non-cancer disease models.

Disease models	Redox activity/Apoptosis regulation	Nanoparticle characteristics	(Refs.)
Alveolar epithelial cells	Antioxidant/Oxidant	Not described	Lord et al (21)
Progressive cochlear and retinal degeneration in Tubby mice	Antioxidant/Apoptosis inhibition	Medium, saline	Kong et al (40)
Hepatic fibrosis	Antioxidant/Apoptosis inhibition	Synthesis, chemical precipitation; Size, 4-20 nm; medium, aqueous solution of TMAOH	Oró et al (41)
Type 1 diabetes	Antioxidant/Apoptosis inhibition	Purchased from Sigma-Aldrich; size, 90 nm; shape, cubes	Khurana et al (42)
Activated and non-activated human monocytic cells	Antioxidant/Oxidant	Synthesis, flame spray pyrolysis; size: 3-94 nm	Schwotzer et al (43)

[i] TMAOH, tetramethylammonium hydroxide; PBS, phosphate saline buffer.

Table II Effects of cerium oxide nanoparticles in cancer cellular models.

Table II

Effects of cerium oxide nanoparticles in cancer cellular models.

Cancer model	Redox activity/Apoptosis regulation	Nanoparticle characteristics	(Refs.)
Breast cancer and healthy breast cells	No effect in breast cancer cells and antioxidant in healthy breast cells	Synthesis, microemulsion process; size, 3-5 nm	Tarnuzzer et al (50)
Lung carcinoma, melanoma and colorectal adenocarcinoma and healthy (origin) cells	Oxidant in tumors/No apoptosis induction	Synthesis, SPRT; shape, cubic; size: 4 nm	Pešić et al (62)
Alveolar adenocarcinoma, hepatoma, colorectal cancer, cervical cancer and healthy (origin) cells	Antioxidant	Purchased from Sigma-Aldrich; size, <25 nm; medium, DMEM	Rubio et al (57)
Hepatoma	Oxidant	Shape, hexahedral; size, 20-30 nm	Cheng et al (58)
Hepatoma	Unchanged/Apoptosis inhibition	Not described	Cheng et al (61)
Hepatoma	Apoptosis induction	Synthesis, chemical precipitation; size, 4-5 nm; medium, aqueous solution of TMAOH	Fernández-Varo et al (60)
Melanoma	Oxidant/Apoptosis induction	Purchased from Sigma- Aldrich; size, 20-40 nm	Ali et al (53)
Melanoma	Oxidant/Apoptosis induction	Purchased from Sciventions; Medium, water	Aplak et al (63)
Colorectal carcinoma	Oxidant/Apoptosis induction	Synthesis, chemical precipitation; size, 30-40 nm	Datta et al (64)

[i] SPRT, self-propagating room temperature method; TMAOH, tetramethylammonium hydroxide.

2. CeO2-x nanoparticles mimic antioxidant enzymes

Due to their physical and chemical redox properties, CeO2-x nanoparticles have begun to be used in the biomedical field, with the aim of restoring normal tissue homeostasis. CeO2-x nanoparticles exhibit controversial pro-oxidant and antioxidant activity, which enable them to react with chemical elements such as oxygen, nitrogen, sulphur and chloride (21). It has previously been described that CeO2-x nanoparticles have the ability to mimic some enzymes, such as catalase (CAT) and superoxide dismutase (SOD), and neutralise reactive oxygen species (ROS) (21).

Korsvik et al (22) have described CeO2-x nanoparticles exhibiting SOD-like activity, hypothesizing that they confer cellular protection. These findings revealed that the surface oxidation state of CeO2-x nanoparticles plays an essential role in the SOD mimetic activity, found to be dependent on the concentration of the +3 oxidation state (22). It suggests a positive association between the trivalent oxidation state of CeO2-x nanoparticles and the mimetic activity of SOD (22,23). A single nanoparticle has been shown to be more efficient as a SOD catalyst than the natural SOD enzyme, with a catalytic rate of 3.6x109 M-1sec-1 compared with 1.3 and 2.8x109 M-1sec-1 of a natural SOD enzyme (22). In contrast to results attributing a SOD mimetic activity for CeO2-x nanoparticles, experiments conducted by Pirmohamed et al (24) showed CeO2-x nanoparticles acting as CAT mimetics in a redox state-dependent manner. Their findings demonstrated that only CeO2-x nanoparticles with fewer surface cerium atoms in the +3 state exhibited any significant CAT mimetic activity. These findings are especially noteworthy, since CeO2-x nanoparticles with lower +3/+4 ratios were revealed to be less efficient in their SOD mimetic activity, and thus there appears to be an inverse realtionship between catalysis and the cerium oxidation state of the nanoparticle (23).

It should be noted that the effects of SOD and CAT mimetics can be enhanced or suppressed by CeO2-x nanoparticle surface modifications. Yadav and Singh (23) demonstrated that CeO2-x nanoparticles coated with phosphotungstic acid (PTA) exhibited an enhanced SOD and CAT mimetic activity independent of the majority of nanoparticle surface charges (+3 or +4). When covering the nanoparticle with phosphomolybdic acid (PMA), SOD activity was suppressed in CeO2-x nanoparticles with its +3 surface charge, however no effect was observed on CAT activity. In addition, PMA covering exerted no effect on SOD while CAT acitvity was enhanced in CeO2-x nanoparticles with a +4 surface charge. PTA and PMA are both electron-dense molecules and display quick and reversible multielectron redox reactions, a probable explanation for the surface charge-dependent effects on SOD/CAT (23).

Notably, a molecule exhibiting the same characteristics as PTA and PMA was demonstrated to exert the opposite effects from those aforementioned. Triethyl phosphite (TEP) altered the SOD and CAT mimetic activities of CeO2-x nanoparticles. A higher Ce4+/Ce3+ surface oxidation state exerted a decrease in CAT mimetic activity while SOD activity was increased. In addition, in this study a correlation between TEP concentration and the formation of surface oxygen vacancies was reported (25).

Of note, PMA, PTA and TEP are molecules that contain phosphorus. For example, Karakoti et al (26) described that CeO2-x nanoparticles coated with the polymer polyethylene glycol (PEG), which does not hold phosphorus in its composition, did not affect the SOD mimicking activity of the nanoparticle. In view of the results herein mentioned, CeO2-x nanoparticles exhibit SOD and CAT mimetic activity, and their effects may be enhanced or suppressed according to which type of molecules cover the the surfaces of the nanoparticles. Further studies analysing the influence of different chemical functional groups on SOD and CAT mimetic activities on the surface of nanoparticles are required.

3. Biological effects of CeO2-x nanoparticles in disease models with dysregulated apoptosis and redox imbalance

Apoptosis is the most studied cell death modality and can be triggered by a wide range of stimuli, such as DNA damage, nutrient deprivation, hypoxia, viral infections, growth factor and hormone signalling. In addition, apoptosis involves the activation of two main biochemical pathways (intrinsic and extrinsic) and leads to various events such as cell retraction, bleb formation, protein cleavage, DNA degradation, and phagocyte recognition (20). Apoptosis dysregulation is involved in the pathogenesis of numerous diseases, including progressive cochlear and retinal degeneration (27), liver fibrosis (28), hypoxia in brain cells (29), type 1 diabetes (30), neurodegenerative diseases (31) and cancer (32). For some of these conditions, apoptosis is defective, and thus cells are capable of surviving even upon cell death stimuli. For others, apoptosis is aberrant, and is associated with organ degeneration and failure. Restoring apoptosis is essential for tissue homeostasis and great effort has been devoted to inducing or inhibiting this form of cell death, depending on the cellular and disease context. Although the scope of this manuscript is apoptosis, the increasingly significant role of other regulated cell death modalities directly modified by cellular oxidative stress, such as ferroptosis or paraptosis, cannot be excluded (20).

Furthermore, the accumulation of ROS has been described to be associated with the development of pathologies such as diabetes, atherosclerosis, stroke, arthrosis, amyotrophic lateral sclerosis, neurodegenerative disorders including Parkinson's and Alzheimer's diseases and cancer (10). ROS play a central role in cell signaling as well as regulation of the main pathways of apoptosis mediated by mitochondria, death receptors and the endoplasmic reticulum (ER) (33-35). Notably, several cytotoxic agents have been shown to induce apoptosis by ROS production (36-39). Nevertheless, low levels of ROS play an important role in the signal transduction process inside the cell, acting as second messengers in the physiological environment. Therefore, it is important to finely tune the balance between cellular oxidative stressors and the antioxidative defence to maintain homeostasis and impair pathological states (10).

In this context, with the increasing interest in the redox characteristics of CeO2-x nanoparticles, numerous authors have dedicated studies to their role in modulating apoptotic signalling pathways in a wide range of disease models.

Effects of CeO2-x nanoparticles in disease models exhibiting excessive apoptosis

Some studies have shown CeO2-x nanoparticles as negatively regulating apoptosis pathways in animal models of diseases that exhibit aberrant apoptosis (Table I; Fig. 2). For example, in an in vivo model of cochlear and retinal degeneration, CeO2-x nanoparticles were shown to upregulate, at the protein level, the basic fibroblast growth factor (bFGF)/receptor tyrosine kinase (RTK)/Ras/extracellular signal-regulated kinase (ERK) pathway, indicated as essential for cell proliferation and survival (40). An increase in the protein expression of thioredoxin (Trx), nuclear factor 2 (Nrf-2), and nuclear Nrf-2 antioxidant proteins and a decrease in the ROS concentration and in the mRNA levels of caspase 8 and BCL-1-antagonist-killer (Bak-1) was also revealed in this study. In addition, a decrease in the catalytic activity of caspases 3 and 9 and improved release of cytochrome c from the mitochondria were reported (40). These findings clearly attribute an antioxidant activity to CeO2-x nanoparticles, at least in the model in context.

Figure 2 Cellular signalling pathways affected by CeO2-x nanoparticles in disease models with excessive apoptosis. In an in vivo model of cochlear and retinal degeneration (40). CeO2-x nanoparticles stimulate the bFGF/RTK/Ras/ERK pathway by reducing ROS. Under this stimulus, the Ras-p-ERK cascade activates Nrf2, which then increases the expression of Trx, further inhibiting the production of ROS. Reduced ROS levels lead to a decrease in the activity of caspases 3, 8 and 9, Bak-1 expression and release of cytochrome c from the mitochondria, inhibiting the apoptosis process. In liver fibrosis, CeO2-x nanoparticles reduce the levels of TNFα, IL1β, COX-2 and iNOS pro-inflammatory cytokines (41). In type 1 diabetes, CeO2-x nanoparticles exhibit a significant decrease in the levels of MDA and an increase in GSH, which may suggest lower ROS levels after treatment with CeO2-x nanoparticles (42). In hypoxic brain cells, CeO2-x nanoparticles coated with polyetilenoglycol (PEG-CeO2-x) act on the AMPK/PKCζ/p-PKCζ/CBP/p-CBP pathway and inhibit caspase-8, leading to neurogenesis and inhibition of the apoptotic process (29). CeO2-x, cerium oxide; FGF, fibroblast growth factor; RTK, receptor tyrosine kinase; ERK, extracellular signal-regulated kinase; ROS, reactive oxygen species; p-, phosphorylated; Nrf2, nuclear factor 2; Trx, thioredoxin; Bak-1, BCL-1-antagonist-killer; TNFα, tumor necrosis factor α; IL1β, interleukin-1β; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; GSH, glutathione; AMPK, 5' AMP-activated protein kinase; PKCζ, protein kinase Cζ; CBP, CREB-binding protein.

For liver fibrosis, a chronic liver disease characterized by excessive apoptosis (28), Oró et al (41) demonstrated that CeO2-x nanoparticles reduced steatosis (accumulation of fat in the liver), portal hypertension (abnormal increase in blood pressure in the portal vein that transposes blood from the intestine to the liver) and the levels of hepatic pro-inflammatory cytokines in rats, thereby attenuating the inflammatory response. Additionally, a marked reduction in the mRNA expression of inflammatory cytokines [tumor necrosis factor α (TNFα), interleukin-1β (IL1β), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)], endothelin 1 (ET-1) and messengers related to the oxidative stress signaling pathway [eosinophil peroxidase precursor (Epx), neutrophil cytosolic factor 1 and 2 (Ncf1 and Ncf2)] or endoplasmic reticulum [cyclic AMP-dependent transcription factor (Atf3) and heat shock protein family A (Hsp70) member 5 (Hspa5)] was observed. This was associated with reduced macrophage infiltration and reduced abundance of active caspase-3 protein, immunostaining of α-smooth muscle actin (α-SMA) and both protein and gene expression of inflammatory cytokines.

An in vivo study conducted by Khurana et al (42) for type 1 diabetes induced in Swiss mice, revealed a significant decrease in malondialdehyde (MDA), a ROS marker, and nitric oxide levels and an increase in glutathione (GSH; part of antioxidant defence) levels and insulin production following CeO2-x nanoparticle treatment. In CeO2-x nanoparticle-treated mice, the levels of intracellular SOD increased while the expression of caspase-3 and DNA damage decreased. Collectively, these findings indicate that CeO2-x nanoparticles act as antioxidant agents and modulate signalling pathways (Fig. 2), particularly those resulting in apoptosis inhibition, in the context of diseases with excessive apoptotic levels.

Conversely, some studies have shown CeO2-x nanoparticles acting as an oxidant agent generating inflammatory and oxidative stress in models exhibiting defective apoptosis, further inducing positively the apoptotic pathway. As an example, a previous study performed by Schwotzer et al (43), revealed CeO2-x nanoparticles acting as inflammatory and oxidative stress agents in in vitro tests in alveolar epithelial cells, verified through the release of chemokines and characterized by the release of increased monocyte activation with subsequent neutrophil and lymphocyte infiltrations. These findings, although in contrast to the previous ones aforementioned, show the duality of CeO2-x nanoparticles and a possible role in the inflammatory system, which remains to be investigated. In cancer, in which cell death is defective (44) mainly due to dysregulation of Bcl-2 family members (45) and inhibitor of apoptosis proteins (46), CeO2-x nanoparticles can also act as an oxidative agent. In the present study, an overview of the effects of CeO2-x nanoparticles in cancer models, from the first published studies to the analysis of how CeO2-x nanoparticles modulate the apoptotic pathway is presented.

Effects of CeO2-x nanoparticles in models exhibiting defective apoptosis: CeO2-x nanoparticles and cancer

It is well known that the development and progression of cancer are associated with adaptation to oxidative stress and dysregulation of the expression of antioxidant enzymes (47,48). In addition, ROS can act as messengers in signal transduction and induce DNA damage, further leading to carcinogenic lesions (49). As described in the previous section, CeO2-x nanoparticles have been shown to be potent scavengers of certain free radicals and may then be potentially effective in diseases exhibiting high levels of ROS production. However, CeO2-x nanoparticles have also been demonstrated to be either protective or induce oxidative stress in cancer, indicating the diversity of the biological effects of CeO2-x nanoparticles in this scenario.

Radioprotective and sensitizing effects of CeO2-x nanoparticles. The first study described in literature assessing the effects of CeO2-x nanoparticles on tumour lines was carried out by Tarnuzzer et al (50), who treated breast carcinoma tumour cells (MCF-7) and breast epithelial cells (CRL8798) with CeO2-x nanoparticles and analysed their response to radiation. While healthy cells were protected by nanoparticles (showing no cytotoxicity), breast tumour cells were more sensitive to radiation. The radioprotective capacity of CeO2-x nanoparticles in healthy epithelial cells could be possibly explained by their antioxidant activity attributed to the chemical characteristic of its self-regenerating redox activity. Conversely, most solid tumours have an acidic microenvironment due to the glycolytic metabolic pathway and exacerbated lactate production (51). Tumour acidosis may then disable the antioxidant activity of the nanoparticle, potentiating their oxidizing activity and, consequently, sensitizing the tumour to radiation therapy (52).

Effect of pH on CeO2-x nanoparticle activity. Variations in pH are important factors affecting CeO2-x nanoparticle activity. In general, neutral pH promotes cytoprotective effects, while acidic pH leads to cytotoxic effects (52). Due to its redox behaviour, CeO2-x nanoparticles can mimic some enzymes such as as SOD, CAT and oxidases (50). When CeO2-x nanoparticles mimic SOD, the dismutation of O2•- and generation of H2O2 and then, O2 is observed. In this case, a higher Ce3+ surface concentration leads the nanoparticles to exert the same mechanism of action as SOD. On the flip side, a higher surface concentration of Ce4+ leads the nanoparticles to mimic CATs, degrading H2O2. In an acidic pH, CAT properties decrease significantly, but SOD properties remain the same. A single CeO2-x nanoparticle has been revealed to be more efficient as a SOD catalyst than the natural enzyme (22,53). Some studies have shown that the CAT-like scavenger activity of CeO2-x nanoparticles is inhibited in an acidic pH environment. Notably, while the rate of superoxide conversion to peroxide is not affected by pH variations, CeO2-x nanoparticles cannot detoxify the hydrogen peroxide at the same rate in an acidic pH. Therefore, CeO2-x nanoparticles could be harmful in a low pH environment (54-56).

A study by Rubio et al (57) identified antioxidant activity of CeO2-x nanoparticles in both human tumoral cell lines and mouse embryonic fibroblast (MEF) non-neoplastic cells. In this study, the influence of pH on the action of CeO2-x nanoparticles in the A549 human lung alveolar adenocarcinoma cell line was assessed, but it was found that the antioxidant properties of CeO2-x nanoparticles were not influenced.

Modulation of the apoptotic pathway by CeO2-x nanoparticles in cancer. A study conducted by Cheng et al (58) revealed that CeO2-x nanoparticles were effective in inducing apoptosis in the SMMC-7721 human hepatoma cell line. In this study, CeO2-x nanoparticles had a non-spherical hexahedral shape between 20-30 nm. After treatment with CeO2-x nanoparticles, increased levels of ROS and MDA marker, and decreased levels of antioxidant SOD, GSH-px and CAT were found (Fig. 3). In addition, there was an increase in the expression of phosphorylated (p)-p38, p-c-Jun N-terminal kinase (JNK) and p-ERK1/2, followed by improved apoptosis rates. According to a study by Xia et al (59), CeO2-x nanoparticles can induce spontaneous ROS production inducing a protective response. This is likely since cellular responses such as inflammation or mitochondrial damage also act as injury response pathways to stressors other than oxidative stress. Upon adaptation, cells can activate the SOD, GSH and CAT antioxidant enzymes.

Figure 3 Cellular signalling pathways affected by CeO2-x nanoparticles in cancer. For human hepatomas, CeO2-x nanoparticles decreased ROS concentration, as shown by the increase in the levels of MDA and decrease in the levels of GSH (58). Under ROS stimuli, expression of MEK/ERK, JNK, p38 and caspase-3 was also increased, leading to activation of an apoptotic cascade. JNK acts on the regulation of Bid and on the inhibition of Bcl-2, affecting the mitochondrial membrane potential and leading to the activation of the apoptotic pathway via caspases. In human melanoma models, CeO2-x nanoparticle treatment promoted an increase in ROS and MDA, but a decrease in GSH, SOD and CAT levels (61). For colorectal carcinoma models, the induction of apoptosis through activation of caspase-3 and caspase-9, release of cytochrome c in the cytoplasm, high ROS levels and disrupted membrane potential (64) is observed. CeO2-x, cerium oxide; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; MEK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SOD, superoxide dismutase; CAT, catalase.

Accordingly, a previous study by Fernández-Varo et al (60) demonstrated that hepatoma cells treated with CeO2-x nanoparticles had an activation of caspase-3, increased expression of ERK1/2 and a decrease in the expression of p-ERK-1/2. Collectively, the results from Cheng et al (58) and Fernández-Varo et al (60) indicate that CeO2-x nanoparticles may activate distinct signalling pathways when triggering apoptosis cell death in hepatoma in vitro models. Conversely, PEG-coated CeO2-x nanoparticles conjugated with alendronate (specific bone resorption inhibitory drug) were found to have protective effects on hepatoma cells, marked by an increased in the ratio of Bcl-2/Bax and p-protein kinase B (p-AKT) and p-ERK, with no change in the levels of ROS (61). In addition, tumour size in vivo increased after CeO2-x nanoparticle treatment, further confirming that CeO2-x nanoparticle-PEG-alendronate promotes cell proliferation and should not be considered for potential therapeutic anticancer purposes.

In human melanoma models, CeO2-x nanoparticles were revealed to have impaired cell viability in a dose-dependent-manner, decreased levels of GSH and increased levels of caspase-3, ROS, MDA and SOD. In addition, there was an increase in double strand DNA breaks following ROS generation, suggesting that CeO2-x may promote oxidative stress-mediated apoptosis and DNA damage (63). Furthermore, in melanoma, Aplak et al (63) found that CeO2-x nanoparticles could induce the levels of mitochondrial ROS, accompanied by an increase in the oxidation of mitochondrial thiol and promotion of hydrogen peroxide-linked mitochondrial dysfunctions. The cytotoxic effects of CeO2-x nanoparticles have also been demonstrated in colorectal carcinoma models, in which a dose-dependent induction of apoptosis was found. This was associated with activation of caspase-3 and caspase-9, release of cytochrome c in the cytoplasm, high ROS levels and disrupted membrane potential (64). Taken together, most preclinical studies indicate that CeO2-x nanoparticles exhibit cytotoxic activity in experimental models of cancers from different origins, which may encourage future work with animal models.

4. Concluding remarks and future perspectives

With the increasing interest in the dual characteristics of CeO2-x nanoparticles, as either an oxidant or antioxidant, numerous authors have begun to investigate their participation in apoptosis signalling pathways. In some cases (as for example, in cancer), its induction would be favourable to help control the disease. In other cases (such as in liver fibrosis), its inhibition would be ideal to interrupt the disease-associated degenerative features.

Apoptosis can be triggered by different factors and several pathways can be activated to promote cell termination. Notably, CeO2-x nanoparticles can modulate some of these pathways, which could be particularly useful for future cancer treatments (44). Although this review highlights the effects of CeO2-x nanoparticles in modulating apoptotic cell death, other forms of cell death have also been shown to be triggered by these nanoparticles (65-67). The cytotoxic effects of CeO2-x nanoparticles in non-neoplastic cells are minor, some even cytoprotective against cytotoxic stimuli.

Considering the dual behaviour of CeO2-x nanoparticles as either antioxidant or pro-oxidant (oxidation no. change between +3 → +4 → +3), it is important to mention that nanoparticle action may not only be influenced by tumour features, but also by its structural and chemical characteristics. It is most likely that the effects of CeO2-x nanoparticles are dependent upon the tumour origin and oxidative state, the subcellular compartment where it is located within cells and how acidic the tumour microenvironment is.

Last but not least, it should be noted that numerous factors such as particle shape, size and structure as well as the solvents used to prepare them for experimental cellular assays may all affect the properties of CeO2-x nanoparticles. Unfortunately, this piece of information is most often overlooked by studies, which lack discussion on the subject. In general, studies have been carried out with nanoparticles of different sizes, synthesis routes and procedures used to prepare their suspension for biological assays. For a better understanding of the effects of CeO2-x nanoparticles in a biological environment, a more detailed description of the chemical structure and properties of nanoparticles must be elucidated (43,54). Combining information from both chemical and biological models is key to address the potential future role of CeO2-x nanoparticles and other nanoparticles in therapeutics and further biomedical applications.

Acknowledgements

Not applicable.

Funding

Funding: The present study was financed in part by the Coordination of Superior Level Staff Improvement-Brazil-(CAPES Finance Code 001), the Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ nos. SEI-26003/004812/2021 and SEI-260003/001554/2022), and the Military Institute of Engineering (IME) and Scholarship for Women in Science L'Oréal-UNESCO-ABC.

Availability of data and materials

Not applicable.

Authors' contributions

All authors (MBDSA, GNDM and KRDS) designed and conceived the study, performed the research, and wrote and edited the manuscript. Data authentication is not applicable. All authors have read and agreed to the published version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References