New insights of cerium oxide nanoparticles in head and neck cancer treatment

Coating and branching characteristics of dextran affect physicochemical properties and stability of Dex-CeNPs

Initial physicochemical characterization experiments which cover the visualization of the color changes during synthesis process, UV-Vis spectra, Dynamic Light Scattering (DLS) and zeta potentials were performed (Fig. 1). In summary, the first indicator of the successful synthesis is the color changes during synthesis process as observed in Fig. 1a²⁴. Each synthesis (SD1 and SD2) was followed up for 18 h. At the end of 1 h, color changes were observed as bright yellow for SD1 and dark brown for SD2 (Fig. 1a, left). In contrast, color changes were observed as dark brown for SD1 and bright yellow for SD2 at the end of the synthesis after 18 h (Fig. 1a, right). The maximum absorbance peak of SD1 and SD2 was observed at 275 and 287 nm respectively via UV-Vis spectra measurements (Fig. 1b). DLS measurements showed that the hydrodynamic diameter of SD1 and SD2 were approximately 10 ± 0.34 nm with 30% ratio and 100 ± 0.4 nm with 35% (Fig. 1c). The zeta potential values of SD1 and SD2 were − 0.029 ± 0.015 and 0.0756 ± 0.110 mV as shown in Fig. 1d. The data indicate us SD1 has slightly negative surface charge where SD2 has a slightly positive surface charge.

(a) Time-dependent color changes during the synthesis of SD1 and SD2 indicate successful synthesis: SD1 transitions from bright yellow to dark brown, while SD2 changes from dark brown to bright yellow after 18 h. (b) UV–Vis spectra (1:100 dilution) reveal maximum absorbance peaks for SD1 and SD2 at 275 and 287 nm, respectively. (c) DLS analysis shows hydrodynamic diameters of SD1 and SD2 as approximately 10 ± 0.34 nm with 30% ratio and 100 ± 0.4 nm with 35% nm. (d) Zeta potential measurements demonstrate a slightly negative surface charge for SD1 and a slightly positive surface charge for SD2.

After initial physiochemical characterization, the Ce³⁺/Ce⁴⁺ ratio of SD1 and SD2 were examined using X-ray photoelectron spectroscopy (XPS). Figure 2a and b showed SD1 and SD2 Ce3d deconvoluted XPS spectra respectively. Ce⁴⁺ peaked at 881.9, 898.0, 900.5, and 906.4 eV, while Ce³⁺ peaked at 885.8 and 903.6. The SD1 contained 66.7% Ce⁴⁺ and 33.3% Ce³⁺ with a Ce³⁺/Ce⁴⁺ ratio of 0.50. SD2 had 59.2% Ce³⁺ and 40.8% Ce⁴⁺ with a Ce³⁺/Ce⁴⁺ ratio of 1.45. As reported in the literature, Li et al. found Ce³⁺/Ce⁴⁺ on CeNPs as 0.80 without the dextran coating²⁵. Costantino et al. observed Ce³⁺/Ce⁴⁺ ratio of 0.42 on cellulose acetate cerium oxide nanoparticle²⁶. The distinctive Ce⁺³/Ce⁺⁴ ratio seen between SD1 and SD2, in contrast to other cerium oxide nanoparticles reported in the literature, can be attributed to variations in their synthesis and coating methodologies. These results provide important insights into the valence states of cerium oxide nanoparticles and highlight the necessity for further investigation into these states’ effects on Dex-CeNPs properties and behavior.

(a,b) XPS spectra reveal distinct Ce³⁺/Ce⁴⁺ ratios for SD1 and SD2, influenced by their synthesis and coating methodologies. (c) HRTEM images of SD1 and SD2 with less than 5 nm core diameters. (d) TGA analysis shows thermal stability profiles of SD1 and SD2, which exhibit an 11% weight reduction in the temperature range of 25–182 °C, attributed to the elimination of adsorbed water from the crystalline lattice of cerium oxide. The more branched structure of SD1 contributes to greater heat resistance compared to SD2.

High resolution transmission electron microscopy (HRTEM) imaging was performed to determine Dex-CeNPs’ core size and lattice structure. Figure 2c shows HRTEM images of SD1 and SD2 nanoparticle homogeneity with less than 5 nm core diameters. SD2 particles were larger than SD1 in Fig. 2c and correlated with DLS data in Fig. 1c. Both syntheses produced lattice fringes with d-spacing of 0.31 nm and 0.20 nm, which match the (111) and (022) planes of fluid catalytic cracking (FCC) CeO₂ (space group of 225). In order to support the HRTEM results, X-ray diffraction (XRD) measurement was performed to analyze crystal structure of the synthesized nanoparticles XRD patterns show the presence of (111), (220), (311), and (331) planes, typical of a FCC crystal (See Supporting Information, Figure S1)²⁷. These results are consistent to each other and in correlation with literature, which shows us FCC structure and homogeneous distribution in solution with, sizes less than 5 nm^14,19. Moreover, Yazici et al., were synthesized 0.1 M (T-10) Dex-CeNPs with a 3–4 nm core size and FCC structure via the same synthesis strategy followed in this study²¹. Similarly, Hanafy et al. reported that the core size of ethylene glycol-coated cerium oxide nanoparticle, formulated using alkaline precipitation method, was less than 5 nm (with an average of 4 nm) and homogeneously distributed²⁸.

Performing thermogravimetric analysis (TGA) in nanoparticle characterization is important to understand its thermal stability, quantification of mass changes, purity, and optimization of synthesis processes. The SD1 and SD2 were tested for thermal stability using TGA from 25 to 825 °C with 10 °C interval per minute. Figure 2d shows TGA graphs of SD1 and SD2. Orange line represents four-stage TGA plots of SD1 decomposition pattern. Weight loss is 13.5% at 25–197 °C, 54% at 204 °C, 72% at 313 °C, and 89.4% at 825 °C in the first four stages. SD2 (blue line) decomposes in three stages: 11% at 25–182 °C, 90% from 182 to 235 °C, and 96.7% at 420–825 °C. SD1 and SD2 decomposition began with water dehydration. SD1 and SD2 lose 11% of their weight between 25 and 182 °C as adsorbed water in cerium oxide’s crystalline structure removed. At a temperature of 225 °C, SD1 lost only about 58% of its weight, while SD2 lost nearly 90%. At 825 °C, SD1 loses 89.4% and SD2 96.7% of its weight. SD1 contains larger and more branched dextran (D1: 9–11 kDa), while SD2 contains a smaller molecular weight and less branched dextran (D2: 6 kDa). The more branched structure makes SD1 more heat-resistant than SD2 (Fig. 2d).

There are various examples in the literature to show functionalization/coating of nanoparticles with different type of polymers effect their decomposition profile by analyzing TGA. For instance, Kaygusuz et al. examined the thermal decomposition profile of functionalized alginate-based cerium oxide nanoparticle using TGA. They found that the functionalized cerium oxide nanoparticle gradually decomposed, losing 88% of its starting mass, while non-functionalized cerium oxide nanoparticle preserved 92% of its initial mass²⁹. Mohammadi et al. found two weight loss stages in double dextran/PEG-coated iron oxide nanoparticles. The degradation of Dextran and PEG occurred in the second stage (220–440 °C) following the evaporation of H₂O molecules³⁰.

Attenuated total reflectance fourier-transform infrared (ATR-FTIR) and Raman spectroscopy were employed to analyze the chemical composition of SD1 and SD2. The ATR-FTIR spectra for synthesis without Ce(NO₃)₃·6H₂O (undoped), each dextran type, and Dex-CeNPs (SD1, SD2) were compared to evaluate the success of synthesis process and understand the doping mechanism, as depicted in Fig. 3a and b. The top graphs (pink lines) of Fig. 3a and b present the ATR-FTIR spectra of two dextran types D1 and D2, respectively. The spectra exhibited similarities, showing the expected bands of IR-sensitive vibration modes. However, the intense OH vibration obscured the aliphatic C–H vibration modes of dextrans, and the C–O–C vibration of the glycosidic bridge between saccharide units was observed at around 1158 cm⁻¹.

(a) ATR-FTIR spectra of [Ce(NO₃)₃·6H₂O, (upper black), D1 (upper pink), SD1 without doping (lower dashed orange), SD1 (lower orange)], (b) [Raman spectra of SD1 and D1], (c) ATR-FTIR spectra of [Ce(NO₃)₃·6H₂O, (upper black), D2 (upper dashed pink), SD2 without doping (lower dashed blue), SD2 (lower blue)], (d) [Raman spectra of SD2 and D2]. ATR-FTIR spectroscopy confirmed the successful dextran coating on cerium oxide, and Raman spectroscopy reinforced these findings by highlighting the high purity of the synthesis process and the effectiveness of the dextran functionalization.

ATR-FTIR spectra of the doping agent Ce(NO₃)₃·6H₂O, also shown in the top graphs (black line) of Fig. 3a and c, revealed characteristic nitrate ion (NO₃⁻) vibration modes as a broad band between 1470 and 1250 cm⁻¹. The bottom orange and blue lines in each graph represented doped SD1 and SD2 respectively, while the dashed orange and blue lines depicted undoped SD1-SD2 samples respectively. The appearance of vibration modes attributed to NO₃⁻ ions and the disappearance of absorption bands between 1000 and 1250 cm⁻¹, attributed to C–O–C vibration in the FTIR spectra, indicated successful doping of SD1 and SD2. This suggests that the cerium anion of Ce(NO₃)₃·6H₂O binds to the oxygen group of the saccharide ring, consistent with similar reaction mechanisms reported in the literature^13,19.

The ATR-FTIR spectra of D1 and D2 used in the synthesis were very similar, with all the expected bands of IR-sensitive vibrational modes observed, as reported by Naha et al.¹⁹. However, the OH vibration, which is intense and has a wide band gap, obscured the C–H vibration. The C–O–C vibration at 1158 cm⁻¹ corresponded to the glycosidic bonds formed between the saccharides. Additionally, a broad band of characteristic nitrate ion (NO₃⁻) vibrational modes was observed between 1470 and 1250 cm⁻¹ in the ATR-FTIR spectra of the doping agent Ce(NO₃)₃·6H₂O. The appearance of the vibrational band attributed to NO₃⁻ ions in the FTIR plots of dextran-coated SD1 and SD2, and the disappearance of the C–O–C resonance band observed in the dextran plot, confirmed the successful coating of both syntheses with dextran. The presence of a peak at 630 cm⁻¹ in the fingerprint region indicated the presence of CeNPs³¹, while the vibration band at 1738 cm⁻¹ was associated with C=O bonds. Peaks of H₂O (H–O–H) molecular bonds were observed at almost the same positions, at 1640 cm⁻¹ for SD1 and 1639 cm⁻¹ for SD2. Furthermore, a weak C–H bending peak at 1467 cm⁻¹ was detected³², along with N–O characteristic vibration bands in the 1340 and 1361 cm⁻¹ region³¹. Consequently, the ATR-FTIR spectroscopy results demonstrated the successful coating of dextran on cerium oxide.

Following the ATR-FTIR experiment, Raman spectroscopy analysis was performed on SD1 and SD2, D1 and D2. The comparison of Raman spectra between SD1 (orange line)/D1 (pink line) and SD2 (blue line)/D2 (dashed pink line) in the spectral region of 100 to 3200 cm⁻¹ is depicted in Fig. 3b and d, respectively. The Raman spectra of D1 and D2 (pink and dashed pink) exhibited characteristic bands throughout the spectrum range. While the spectral patterns of SD1 and SD2 were similar, the SD2 (blue line) spectrum revealed a new band at 714 cm⁻¹ (Fig. 3d). Additionally, the band observed at 1036 cm⁻¹ in SD1 shifted to 1050 cm⁻¹ in SD2 as shown in Fig. 3d. Both SD1 and SD2 lacked the characteristic Raman band at about 464 cm⁻¹, attributed to the symmetrical stretching mode of Ce–O vibration, indicating successful coating of CeO₂ with dextran. Notably, no fingerprint band for CeO₂ at 464 cm⁻¹ was observed in either synthesis (SD1 and SD2), confirming the presence of dextran coating³³. The Raman spectroscopy analysis results supported the ATR-FTIR data and demonstrated the purity of the syntheses and the successful dextran coating.

Following detail characterization experiment; age dependent changes, which are influenced by various factors within the microenvironment such as light, radiation, moisture, pH, temperature, ionic strength, and concentration³⁴ were questioned. Therefore, the stability of SD1 and SD2 nanoparticles was analyzed over 6 months at different time intervals using UV-Vis and DLS measurements (see supporting information). 28 days as a critical time period was observed for both syntheses. The change in DLS and UV measurements show us SD2 is more stable than SD1 for over the course of 6 months.

The characterization data except Raman, ATR-FTIR, HRTEM, and XRD was summarized in Table 1 to address the difference between the physicochemical properties of SD1 and SD2. Raman and ATR-FTIR data indicate us successful synthesis of SD1 and SD2 through dextran coating. HRTEM and XRD (in supporting information) data shows the same crystalline structure with very close core size in correlation.

In summary, the more branched structure of the dextran (D1) used for SD1 makes the thinner and entangled coating on cerium oxide. On the other hand, the dextran (D2) used for SD2, which has less and longer branched structure, makes the coating on the cerium oxide thicker and larger. The thinner coating characteristics of SD1 gives smaller absorbance maxima at 275 nm. In contrast, SD2 with thicker coating, bigger size gives absorbance maxima at 287 nm. The OH– groups are in more contact with cerium core for SD1 due to entangled coating, which makes its surface charge slightly positive. These groups are more available on the surface due to disentangled coating for SD2, which makes its surface charge slightly negative. These coating properties of Dex-CeNPs cause different heat stability as such higher for SD1 and lower for SD2. Besides, SD1, with more bounds on the surface, is more prone to change and less stable, whereas SD2, with fewer bounds, is less prone to change and therefore more stable in terms of size.

Dex-CeNPs with no mutagenic potential in the AMES test

Evaluating the genotoxicological profile is necessary to assess the use of any nanoparticles in clinics. This profile can be modulated by physicochemical characteristics of nanoparticles (e.g., size and coating) which also directs internalization of them regarding cellular uptake³⁵. Therefore, the genotoxicity profile of Dex-CeNPs were evaluated via Ames test performed with five S. typhimurium strains. Since there was no two-fold or more increase in revertant colonies of samples compared to spontaneous and negative control the AMES test of the samples was considered as negative (−) result. It was observed that “SD1 and SD2” are non-mutagenic in these test conditions and in the tested S. typhimurium strains (see supporting information)

Characteristic cytotoxicity and ROS profiles of Dex-CeNPs across HNC cell lines temporal and dose-dependent cytotoxicity of Dex-CeNPs in HNC cell lines

The potential of Cerium oxide nanoparticle as an anti-cancer agent has been studied extensively in breast, pancreas, ovarian, lung, colon, and bone cancer cells through various surface modifications^21,36,37. However, its effectiveness in treating HNC, a challenging cancer type, remains unclear so far. In this study, SD1 and SD2 were tested against HNC cell lines (A253, FaDu, SCC-25) to assess their cytotoxicity profile. These three cell lines were chosen based on their anatomical locations and the prevalent mutations as initial criteria (see supporting information). Specifically, The FaDu cell line is from the pharynx, A253 from the salivary gland, and SCC-25 from the tongue. Among these prevalent mutations TP53, regarded as the guardian of genome, is a pivotal determinant in assessing the effectiveness of various cancer treatment strategies and the most common genetic alteration in head and neck cancer (HNC), occurring in roughly 70% of cases. The molecular profiles of the selected cell lines and their anatomical regions were comparatively given in the supporting information as Table S1.

Initially, the cytotoxicity was evaluated in a dose and time-dependent manner (on Day 1 and on Day 3) for each cell line. In A253 cells, a dose-dependent change was observed after SD1 and SD2 treatments, whereas time dependence was not significant as shown in Fig. 4a. At 100 µg/mL, SD1 and SD2 had no significant effect on A253 cell viability. However, at 250 µg/mL, SD2 had reduced cell viability by more than 50% on both Day 1 and Day 3, while SD1 had approximately 20% cytotoxicity effect. At higher concentrations (500 and 1000 µg/mL), there is no remarkable change in terms of cell viability in the presence of SD1 and SD2 for both days. In FaDu cells, SD1 and SD2 had different cytotoxicity trend as shown in Fig. 4b. At a concentration of 100 µg/mL, no effect was observed on Day1, while approximately 40% cytotoxicity was observed on Day 3 for SD1. A similar result appears at the concentration of 250 µg/mL. At a concentration of 500 µg/mL, approximately 20% cytotoxicity was observed on Day 1, and 60% cytotoxicity was observed on Day 3. An increased cytotoxic effect (30%) was observed at 1000 µg/mL concentration on Day 1 compare to 500 µg/mL, while a similar trend with 500 µg/mL was observed on Day 3.

Cytotoxic properties of SD1 and SD2 against (a) A253, (b) FaDu, (c) SCC-25 cell lines at 100, 250, 500, 1000 μg/mL concentrations after 1 and 3 days of treatment. SD1 and SD2 displayed time and dose-dependent variability across cell lines. *Represents comparison between SD1/SD2 Day 1 and control group for each concentration (p value ≤ 0.05). ^#Represents comparison between SD1/SD2 Day 3 and control group for each concentration (p value ≤ 0.05). **Represents comparison between SD1, Day 1 and SD2, Day 1 at the same concentration (p value ≤ 0.05). ^##Represents comparison between SD1, Day 3 and SD2, Day 3 at the same concentration (p value ≤ 0.05). ROS generation of SD1 and SD2 against (d) A253, (e) FaDu, (f) SCC-25 cell lines at 100, 250, 500, 1000 μg/mL concentrations after 24H of treatment. The variation in ROS production depend on applied dose and the Ce³⁺/Ce⁴⁺ ratio between SD1 and SD2. *Represents comparison between SD1 control group and each concentration (p value ≤ 0.001). ^#Represents comparison between SD2 control group and each concentration (p value ≤ 0.001). **Represents comparison between SD1 and SD2 at the same concentration (p value ≤ 0.001).

On the other hand, SD2 showed very little cytotoxic effect on FaDu cell line up to 500 µg/mL on both Day 1 and Day 3, indicating inefficient long-time effect compared to SD1. At a concentration of 500 µg/mL, SD2 resulted in 40% cytotoxicity on Day 1 and 20% on Day 3. At 1000 µg/mL, SD2 exhibited 70% cytotoxicity on Day 1 and 30% on Day 3 (Fig. 4b). It has been observed that SD1 is much more cytotoxic in long-term exposure compare to SD2, while SD2 was more cytotoxic after Day 1 treatment. It is plausible that the distinct pharmacokinetic properties and temporal dynamics of SD2, in conjunction with the complex cellular microenvironment, may contribute to this temporal shift in cell viability³⁸.

SD1 and SD2 treated SCC-25 cells showed no significant effect at 100 µg/mL on both Day 1 and Day 3. However, at 250 µg/mL, 500 µg/mL and 1000 µg/mL, SD2 treatment reduced cell viability by about 75% on Day 1. Moreover, cytotoxicity effect was around 80% at 250 µg/mL, and 90% was observed at 500 µg/mL and 1000 µg/mL on Day 3 while SD1 had no significant impact over both days for all concentrations (Fig. 4c).

Depending on cytotoxicity profile observed and reported above, Half-maximal inhibitory concentration (IC₅₀) were calculated as indicated in Table 2. SD1 has IC₅₀ (µg/mL) values for A253 (315), FaDu (347), SCC-25 (> 1000), SD2 has IC₅₀ (µg/mL) values for A253 (129), FaDu (292), SCC-25 (225).

Evaluation of SD1 in terms of time dependent manner shows us no difference in cell viability for A253, SCC-25 and higher cytotoxic effect over long term exposure for FaDu. The variation in time dependent efficacy of SD2, where exhibited no daily basis cell viability difference for A253; more cytotoxic effect on Day 1 compare to Day 3 for FaDu; slightly less cytotoxic effect on Day 1 compare to Day 3 for SCC-25 was observed.

The change in effectiveness over time of Dex-CeNPs against HNC cell lines can be attributed to the complex dynamics of drug such as initial uptake, cell adaptation, and non-linear dose–response relationships. Moreover, these findings underscore the multifaceted nature of drug–cell interactions and highlight the need for a comprehensive understanding of these factors during the evaluation of potential drug candidates³⁹.

Ce³⁺/Ce⁴⁺ ratio as a key driver of ROS generation of Dex-CeNPs in HNC cell lines

Following, we focused on analyzing ROS generation properties of SD1 and SD2 on HNC cell lines. Figure 4d–f display the ROS fold change after one day of SD1 and SD2 treatment on HNC cells.

In A253 cells (Fig. 4d), ROS production at 100 and 250 µg/mL of SD1 was the same with control group, while 500 µg/mL increased it by 1.5-fold. It was increased around 3-folds at 1000 µg/mL compare to control. For SD2, no fold difference at 100 µg/mL, 3-folds at 250 µg/mL, 2-folds at 500 µg/mL, 1.5-fold difference at 1000 µg/mL compared to control group were observed in terms of ROS generation. ROS enhancement was negligible for SD1 and SD2 around the IC₅₀ concentration (315 µg/mL and129 µg/mL respectively) compare to control.

In FaDu cells (Fig. 4e), SD1 had slightly increased ROS production at 100 and 250 µg/mL compared to the control group. This increase escalated to 1.7-fold at 500 µg/mL, and 2-folds at 1000 µg/mL. Conversely, SD2 demonstrate 1.5 fold (100 and 250 µg/mL) and exceeding 1.5-fold across all concentrations on FaDu cells. SD2, at all concentrations, had an impact of more than 1.5-fold on FaDu cells. The IC₅₀ values for FaDu cells were 347 µg/mL for SD1 and 292 µg/mL for SD2. Compared to A253 cells, FaDu cells generated more ROS at concentrations around the IC₅₀.

For the SCC-25 cell line (Fig. 4f), SD1 showed no significant impact on ROS generation at all concentrations, while SD2 elicited notable effects with four-folds, six-folds, and five-folds increase in ROS generation at 250, 500, and 1000 µg/mL, respectively. The IC₅₀ values for SCC-25 cells were > 1000 µg/mL for SD1 and 225 µg/mL for SD2. Non-signifcant ROS production was observed for SD1 while SD2 showed more than four-folds increase at around IC₅₀ concentration for each nanoparticle.

ROS generation for cerium oxide NPs, as nanozymes, is related with the Ce³⁺/Ce⁴⁺ ratio on the nanoparticle surface which also defines its mimetic activity of redox enzymes such as SOD, catalase, phosphatase, oxidase peroxidase, and phosphotriesterase. The Ce³⁺/Ce⁴⁺ ratio, with Ce³⁺ dominated ratio exhibiting stronger SOD-like activity and with dominated Ce⁴⁺ ratio have catalase and phosphatase-mimetic activities⁴⁰.

In this study, SD1 has a Ce³⁺/Ce⁴⁺ ratio of 0.5, while SD2 has a ratio of 1.45, depending on XPS data. This data gives us, SD2 may have a role as superoxide dismutase, whereas SD1 mimics catalase. The correlation between the Ce³⁺/Ce⁴⁺ ratio of Dex-CeNPs and ROS generation may change its function though enzymatic behavior as we observed in this study^41,42.

A higher potency and improved efficacy of SD2 as an anti-cancer agent, our subsequent decision to focus on SD2 for further experiments using flow cytometry analysis and gene expression profiling.

Divergent apoptotic and necrotic responses to Dex-CeNPs (SD2) in HNC Cell lines revealed by flow cytometry

Apoptosis, which initiated by 2 main pathways: intrinsic and extrinsic (controlled by pro- and anti-apoptotic regulatory proteins of the Bcl-2 family and Caspases), is a part of the normal physiological process that ensures tissue homeostasis. The regulation of apoptosis was damaged in cancerous cells, which develop “Resistance to cell death”⁴³. In the content of this study, Flow Cytometry Analysis and gene expression profiling were performed to understand the apoptotic effect of SD2 in the following sections.

HNC cell line apoptosis profile following SD2 treatment was evaluated with Annexin V/PI double immunostaining. Three different concentrations (IC₅₀, below the IC₅₀ and above the IC₅₀ which overlaps with the cell viability data) were chosen for flow cytometry analyses in the presence of three cell lines.

Figure 5a illustrates the healthy profile of untreated A253 cells, showing 88.7% viability (Fig. 5e). At a concentration of 100 µg/mL (below IC₅₀), the viability dropped to 16.21% ± 0.33%, (p < 0.001) (Fig. 5b,e), at IC₅₀-130 µg/mL it fells to 5.39% ± 0.52%, (p < 0.001) (Fig. 5c,e), and at 250 µg/mL (above IC₅₀), it was reduced to 7.04% ± 0.98%, (p < 0.001) (Fig. 5d,e).

SD2 predominantly induces late apoptosis in A253 cells at IC₅₀ concentration. A253 cells were left (a) untreated (negative control) or treated for 24 h with SD2 at (b) 100 μg/mL, (c) IC₅₀-130 μg/mL, (d) 250 μg/mL. Results are expressed as the percentage of cells corresponding to Q1: early apoptotic cells (Annexin V⁺/PI⁻); Q2: late apoptotic cells (Annexin V⁺/PI⁺); Q3: necrotic cells (Annexin V⁻/PI⁺); and Q4: healthy. Cells (Annexin V⁻/PI⁻). Red dot blots (SSC-A/FSC-A) represent corresponding backgate. Representative example of 5 independent experiments. (e) The table refers to the percentage of cells corresponding to each apoptotic level (Mean ± SD). Two-way ANOVA test with Tukey correction across each row indicates statistical significance *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Interestingly, while there is no significant increase of early apoptotic level at 100 µg/mL and IC₅₀-130 µg/mL concentration (5.23% ± 0.72%, 6.84% ± 0.23% respectively, p = ns), compared to 250 µg/mL (50.6% ± 1.27%, p < 0.001). A direct switch to late apoptotic state is detected in lower IC₅₀ and IC₅₀ conditions (58.2% ± 0.42%, 63.35% ± 4.81% respectively, p < 0.001). In 100 µg/mL and IC₅₀-130 µg/mL of SD2, necrotic cells amount are higher than 250 (12% ± 1.07%, p < 0.001). These reductions indicate that SD2 induces late apoptosis at IC₅₀.

Figure 6 shows FaDu cell apoptosis profile after SD2 treatment. In all situations, cell amount in early and late apoptosis increase in dose-dependent manner. The necrotic quantities were the same for 250 and 500 µg/mL, while this amount increased at the IC₅₀-290 µg/mL concentration (40% ± 6.65%).

SD2 Predominantly induces late apoptosis in FaDu cells at IC₅₀ concentration. FaDu cells were left (a) untreated (negative control) or treated for 24 h with SD2 at (b) 250 μg/mL, (c) IC₅₀-290 μg/mL, (d) 500 μg/mL. Results are expressed as the percentage of cells corresponding to Q1: early apoptotic cells (Annexin V⁺/PI⁻); Q2: late apoptotic cells (Annexin V⁺/PI⁺); Q3: necrotic cells (Annexin V⁻/PI⁺); and Q4: healthy. Cells (Annexin V⁻/PI⁻). Red dot blots (SSC-A/FSC-A) represent corresponding backgate. Representative example of 5 independent experiments. (e) The table refers to the percentage of cells corresponding to each apoptotic level (Mean ± SD). Two-way ANOVA test with Tukey correction across each row indicates statistical significance *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

SCC-25 cells exhibit heightened early and late apoptosis percentage in dose-dependent manner like FaDu cells (Fig. 7). Only IC₅₀-220 µg/mL concentration causes considerable necrosis amount in SCC-25 cells (9.61% ± 1.69%, p < 0.001).

SD2 predominantly induces late apoptosis in SCC-25 cells at IC₅₀ concentration. SCC-25 cells were left (a) untreated (negative control) or treated for 24H with SD2 at (b) 100 μg/mL, (c) IC₅₀-220 μg/mL, (d) 250 μg/mL. Results are expressed as the percentage of cells corresponding to Q1: early apoptotic cells (Annexin V⁺/PI⁻); Q2: late apoptotic cells (Annexin V⁺/PI⁺); Q3: necrotic cells (Annexin V⁻/PI⁺); and Q4: healthy. Cells (Annexin V⁻/PI⁻). Red dot blots (SSC-A/FSC-A) represent corresponding backgate. Representative example of 5 independent experiments. (e) The table refers to the percentage of cells corresponding to each apoptotic level (Mean ± SD). Two-way ANOVA test with Tukey correction across each row indicates statistical significance *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

According to the data presented, although all three cell lines were sensitive to SD2, their response was not necessarily dose-dependent. With below IC₅₀ and IC₅₀ concentrations of SD2, A253 cells skip early apoptosis and go straight to late stage. Unexpectedly, the highest dose of SD2 induces early apoptosis but not late or necrosis. The IC₅₀ concentration had the greatest apoptotic effect on A253 and SCC-25 cell lines. In FaDu and SCC-25 cell lines, SD2 at the below IC₅₀ and above IC₅₀ concentrations has a milder necrotic effect than IC₅₀. While the highest necrotic cell amount was observed at below IC₅₀ concentrations for A253.

The divergence in cellular responses observed in A253, SCC-25, and FaDu cell lines, A253 and SCC-25 displaying late apoptosis and FaDu undergoing necrosis upon interaction with IC₅₀ concentration of SD2, can be attributed to a multitude of factors. This response could potentially be assign to variations in the cellular signaling pathways and inherent molecular profiles specific to each cell line. This discrepancy in the mode of cell death may arise from differences in the expression levels of key regulatory proteins involved in apoptotic and necrotic pathways, which ultimately dictate the cellular response to external stimuli such as SD2^44,45.

Flow cytometry shows the basic apoptotic profile of SD2-treated cell lines. These cell specific apoptotic profiles demonstrated in the flow cytometry analysis have led us to explore the apoptosis related gene expression profile.

Differential regulation of apoptosis-related genes by Dex-CeNPs (SD2) in HNC cell lines through RT-PCR analysis

In the present study, the expression level of pro-apoptotic (BAX, CASP3) and anti-apoptotic (Bcl-2) genes were examined in A253, FaDu and SCC25 treated with SD2 IC₅₀ amount for each cell line. In addition, changes in the expression level of TP53, which is mutated in all cell lines and a key factor in the decision of DNA repair or apoptosis of the damaged cell, were also analyzed. The disparities in gene expression levels were quantified as fold changes between treated and untreated (control group) conditions, as illustrated in Fig. 8.

(a,b) Upregulation of the pro-apoptotic genes (BAX), along with CASP3 and TP53, and downregulation of the anti-apoptotic gene (Bcl-2), demonstrating the apoptotic response induced by SD2 treatment in A253 and FaDu cells, respectively. (c) In the SCC-25 cell line, expression levels of all genes (BAX, CASP3, TP53, and Bcl-2) were increased following SD2 treatment at IC₅₀ concentration. Signal intensities of relative genes are normalized to β-actin gene expression level. Quantitative results are presented as the mean ± SD from three independent experiments. Statistical significance was evaluated with Student t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

In Fig. 8a, a notable upregulation in the expression of pro-apoptotic genes was observed in A253 cell line treated with SD2: CASP3 increased by approximately 2-folds, while BAX exhibited a ~ 1.4-fold increase. Conversely, the expression of the anti-apoptotic Bcl-2 gene was decreased by approximately 0.6-fold. Additionally, there was a ~ 1.2-fold increase in the expression of the TP53 gene.

As demonstrated in Fig. 8b, FaDu cell line treatment boosted pro-apoptotic CASP3, BAX and TP53 gene expression by 1.3, 1.2, and 1.5 fold, respectively, while decreasing Bcl-2 gene expression by 0.3-fold.

When the effect of SD2 synthesis was compared with the control group in the SCC-25 cell line in Fig. 8c, an increase in the expression level of all the genes was observed. The gene expression profiles of CASP3 gene (~ 2-fold), BAX gene (~ 1.6-fold) and TP53 gene (~ 1.8-fold) showed similar increasing trends consistent with other cell lines. However, the expression level of anti-apoptotic Bcl-2 gene, which has decreased gene expression level in other cell lines, increased ~ 1.8-fold in SCC-25 cell line.

As we described above, the expression levels of the TP53 and CASP3 genes considerably increased in all three cell lines after treatment of SD2. The CASP3 gene encodes a homonymous protein that acts as an effector caspase in both the extrinsic and intrinsic pathways⁴⁶. This protein induces nucleases and proteases, causing lysis of the cell. CASP3 causes DNA damage by activating CAD-i, which causes DNA breaks in the nucleus. This effect is transmitted via the TP53 signaling pathway. The TP53 signaling pathway is known to act as an inducer in the extrinsic pathway of apoptosis. However, p53 protein, which is the product of the over-expressed TP53 gene, is localized to mitochondria and causes the initiation of apoptosis by activating BAX⁴⁷.

According to the RT-PCR results, the expression levels of the three defined pro-apoptotic genes were increased, providing further evidence for the apoptotic effect of SD2. The overexpression of pro-apoptotic genes reveal that the expression of Bcl-2 as an anti-apoptotic gene, will be reduced. Unexpectedly, in the SCC-25 cell line, Bcl-2 expression increased despite the apoptotic effect of SD2. However, BAX expression also increased in this cell line, even though Bcl-2 typically suppresses BAX. This unexpected result may explain effect of BH3 proteins on over expression of BAX^48,49.

FACS results showing that cells treated with SD2 at IC₅₀ compared to the control group went to apoptosis for A253 and SCC-25. On the other hand, a high proportion of cells treated with IC₅₀ of SD2 were observed in the necrosis stage for FaDu. In the case of A253, increased in expression level of pro-apoptotic genes (CASP3 and BAX) and decrease in anti-apoptotic gene (Bcl-2) was statistically significant, which is in correlation with FACS results. In the case of FaDu, we have the same expression profile with A253, but it was not that much statistically significant. Even though we have a certain amount of cells in apoptosis stage, most of the cells entered necrosis, which supports FACS data. In the subject of SCC25 cells, we have the similar FACS profile with A253 in terms of cell amounts in apoptosis stage. Moreover, statistically significant expression change in pro-apoptotic genes (CASP3 and BAX) were observed for SCC25, which supports FACS results.