Bioactive glass nanoparticles induce strong preferential cytotoxicity and excessive ROS-mediated oxidative stress and apoptotic genomic DNA damage in non-small lung cancer cells
Hanan R. H. Mohamed, Amira H. Yehia
TL;DR
Bioactive glass nanoparticles show strong selective toxicity against non-small lung cancer cells by causing oxidative stress and DNA damage, outperforming doxorubicin.
Contribution
Demonstrates that bioactive glass nanoparticles have superior selective cytotoxicity and induce stronger oxidative stress and DNA damage in NSCLC cells compared to doxorubicin.
Findings
BGNPs show a high selectivity index of 124.31 with no toxicity to normal fibroblasts.
BGNPs induce greater oxidative stress, DNA damage, and apoptosis than doxorubicin in A549 cells.
BGNPs modulate apoptosis-related genes like p53, Bax, and Bcl2 more significantly than doxorubicin.
Abstract
Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and remains the leading cause of cancer-related mortality worldwide. The limited efficacy and high toxicity of current treatment strategies, including chemotherapeutics like doxorubicin, underscore the urgent need for safer, more selective anticancer strategies. Bioactive glass nanoparticles (BGNPs), commonly used for bone regeneration and antimicrobial applications, have recently gained attention for their potential anticancer properties. However, their effects on lung cancer cells, particularly NSCLC, are still not fully understood. The present study consequently was conducted to estimate the therapeutic potential of BGNPs against A549 NSCLC cells. Our findings revealed that BGNPs exert potent, concentration-dependent targeted cytotoxicity toward A549 cancer cells, with a remarkably high selectivity index of…
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Figure 9- —Faculty of science Cairo university
- —Cairo University
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TopicsBone Tissue Engineering Materials · Management of metastatic bone disease · Bone health and treatments
Introduction
Lung cancer is one of the most commonly diagnosed malignancies and remains a leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of cases (Duma et al. 2019; Sung et al. 2021). Among NSCLC subtypes, lung adenocarcinoma is the most prevalent subtype, and the A549 cell line derived from a human alveolar basal epithelial adenocarcinoma is widely used as an in vitro model for preclinical studies (Korrodi-Gregório et al. 2016; Hynds et al. 2021). Despite significant advances in treatment approaches, including surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the 5-year survival rate for advanced-stage NSCLC remains below 20%, largely due to late diagnosis, therapeutic resistance, and systemic toxicity associated with current modalities (Chen et al. 2015; Hirsch et al. 2017).
Chemotherapy, particularly platinum-based regimens such as cisplatin and paclitaxel, remains a cornerstone of NSCLC treatment. However, these drugs lack specificity for cancer cells and indiscriminately target rapidly dividing cells, causing collateral damage to healthy tissues. Consequently, patients frequently suffer from severe dose-limiting toxicities, including nephrotoxicity, neurotoxicity, myelosuppression, and gastrointestinal complications (Zitvogel et al. 2008; Yang et al. 2022). These adverse effects not only reduce quality of life but also limit the therapeutic window of chemotherapy. Such challenges underscore the pressing need for more selective, targeted, and biocompatible therapeutic strategies that can effectively eliminate tumor cells while minimizing harm to normal tissues, thereby improving both the safety and efficacy of lung cancer treatment.
Nanomedicine is an emerging and rapidly advancing field in cancer therapy, offering solutions to many limitations of conventional treatments. By enabling targeted drug delivery, controlled release, and selective accumulation at tumor sites, nanomedicine can enhance therapeutic efficacy while reducing systemic toxicity (Jain and Stylianopoulos 2010; Shi et al. 2017). Among the various nanomaterials explored, bioactive glass nanoparticles (BGNPs) have attracted growing attention due to their excellent biocompatibility, adjustable surface characteristics, and intrinsic biological activity (Cannio et al. 2021; Drevet et al. 2024). Originally, BGNPs were developed for bone regeneration and dental applications and consist primarily of silicon, calcium, sodium, and phosphate ions in an amorphous glass matrix. In physiological conditions, BGNPs undergo controlled degradation, releasing therapeutic ions over time (Hoppe et al. 2011; Jones 2013). These released ions have been shown to modulate cellular signaling pathways involved in proliferation, apoptosis, and oxidative stress, thereby extending the potential use of BGNPs into the realm of cancer therapy (Pajares-Chamorro and Chatzistavrou 2020).
In oncology, recent studies have shown that bioactive glass, in bulk or macroparticulate forms, can exhibit selective cytotoxicity toward cancer cells. For example, Deliormanlı et al. (2024) demonstrated that borate-based bioactive glass particles significantly inhibited the proliferation of human osteosarcoma and glioblastoma cells, while exerting minimal effects on healthy stromal cells. Similarly, Fellenberg et al. (2022) found that macroparticulate bioactive glass triggered mitochondrial dysfunction and apoptosis in bone tumor cells through calcium-mediated signaling and reactive oxygen species (ROS) generation. However, the biological effects of bioactive glass at the nanoscale may differ substantially from their bulk counterparts due to enhanced surface area, increased cellular uptake, altered ion dissolution kinetics, and increased surface reactivity (Cannio et al. 2021). These nanoscale properties may enable BGNPs to exert more potent and selective cytotoxic effects on malignant cells compared to bioactive glass macroparticles, highlighting their potential as an advanced therapeutic platform in cancer treatment.
Despite these promising attributes, there is a notable lack of data on the cytotoxic potential of BGNPs in lung cancer, particularly in NSCLC models. Most available studies have focused on the regenerative and antimicrobial properties of BGNPs, with limited exploration of their direct effects on cancer cell viability, genomic stability, or mitochondrial function (Kaou et al. 2023). Furthermore, the mechanisms underlying BGNPs-induced cytotoxicity, such as oxidative stress, mitochondrial membrane potential disruption, DNA damage, and transcriptional modulation of apoptotic genes, have not been systematically explored in lung cancer cells. Given the distinct physicochemical behavior of nanoparticles and their enhanced interactions with intracellular structures, it is scientifically unsound to extrapolate data from bulk or macroparticle-based systems to the BGNPs without direct empirical validation (Cannio et al. 2021; Drevet et al. 2024).
To address this critical knowledge gap, the present study aimed to estimate, for the first time, the intrinsic cytotoxic potential of BGNPs on A549 human NSCLC cells, independent of any chemotherapeutic drug loading. To evaluate BGNPs selectivity and safety, viability of normal human skin fibroblasts (HSF) was also estimated. This study also evaluated multiple hallmarks of cellular and DNA injury in A549 cancer cells including genomic DNA integrity, intracellular ROS generation level, mitochondrial membrane potential, and expression level of apoptosis- and mitochondria-related gene markers (p53, Bcl-2, and ND3 genes, respectively). By elucidating the multifaceted mechanisms underlying BGNPs-induced cytotoxicity, spanning oxidative stress, mitochondrial dysfunction, DNA damage, and gene expression changes, this study provides novel insights into the anticancer potential of BGNPs in NSCLC. These findings support the development of BGNPs as standalone nanotherapeutics with improved tumor specificity and reduced off-target toxicity, paving the way for future translational and in vivo research.
Materials and methods
BGNPs and reagents
The BGNPs used in this study were obtained as a fine white powder from Nanotech Company (6th October City, Cairo, Egypt). For experimental preparation, the BGNPs were dispersed in dimethyl sulfoxide (DMSO; CAS No. 67–68-5; Sigma-Aldrich, St. Louis, MO, USA) to prepare stock solutions at the desired concentrations. Prior to use, the suspensions were ultrasonicated in a bath ultra-sonicator for 15–20 min to ensure homogeneous dispersion and minimize particle agglomeration. Other analytical and molecular-grade reagents utilized throughout the study, such as 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT) and trypan blue dye, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture reagents, including Dulbecco’s Modified Eagle Medium (DMEM), HEPES buffer, L-glutamine, gentamycin, and 0.25% Trypsin–EDTA, were obtained from Lonza (Belgium). The media were supplemented with 10% fetal bovine serum (FBS) and 1% gentamycin to support cell growth and maintain sterility. All culture media were phenol red-free to avoid interference with spectrophotometric analyses. Reagents were freshly prepared as needed, and all cell culture and treatment procedures were performed under aseptic conditions in a Class II laminar flow biosafety cabinet to ensure sterility and experimental reproducibility.
Characterization of BGNPs
The BGNPs utilized in this study were well characterized to confirm their structural, morphological, and colloidal properties. X-ray diffraction (XRD) analysis was performed using an XPERT-PRO diffractometer (PANalytical, Almelo, Netherlands) with Cu Kα radiation to determine the crystalline or amorphous nature of the used nanoparticles by identifying characteristic diffraction patterns. Transmission electron microscopy (TEM) was carried out on a Tecnai G20 Super Twin microscope (FEI, USA) operating at 200 kV. For TEM, a drop of BGNPs suspension was placed on a carbon-coated copper grid and air-dried to visualize particle shape, size, and aggregation.
Culture of normal HSF and cancerous A549 cells
Human non-small cell lung cancer (A549) and normal skin fibroblast (HSF) cells were obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Both cell Lines were cultured in high-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml streptomycin, and 100 units/ml penicillin to support cell growth and prevent bacterial contamination. The cells were maintained in a humidified incubator at 37 °C with 5% carbon dioxide (CO₂) to simulate physiological conditions.
Evaluation of BGNPs effect on HSF and A549 cell viability
The effect of BGNPs on the viability of normal HSF and cancer A549 cells was assessed using the MTT assay, as described by Mosmann (1983) and Ahmed et al. (2022). Briefly, 100-µl aliquots of cell suspensions containing 5 × 10^3 cells were seeded into 96-well plates and allowed to adhere in complete culture medium for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. After the initial incubation, cells were treated with 100 µl of fresh media containing BGNPs at varying concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μg/ml) and incubated for an additional 48 h under the same conditions. Following treatment, the medium was carefully removed and replaced with 100 µl of phosphate-buffered saline (PBS) containing 20 µl of MTT solution (1 mg/ml). The plates were incubated for 4 h at 37 °C to allow formazan crystals to form, which were then solubilized by adding 100 µl of absolute DMSO to each well. The absorbance of the resulting formazan solution was measured at 570 nm using a microplate reader (BMG LABTECH FLUOstar Omega, Germany). The half-maximal inhibitory concentration (IC50) values were determined using non-linear regression analysis in GraphPad Prism software (San Diego, CA, USA), based on results from three independent experiments. To assess BGNPs’ selectivity, the Selectivity Index (SI) was calculated as the ratio of the IC50 value in normal HSF cells to that in A549 cancer cells. All results are presented as mean ± standard deviation (SD).
Treatment schedule of A549 lung cancer cells
Human A549 lung cancer cells were seeded into T25 flasks and cultured under optimized conditions to promote healthy growth. The cells were maintained in standard culture medium supplemented with FBS, antibiotics, and essential nutrients, at 37 °C in a humidified atmosphere with 5% CO₂. The medium’s pH was carefully controlled within the physiological range. Upon reaching suitable confluency, the A549 cells were divided into untreated and treated cells. Control cells received less than 0.1% DMSO, while treated cells were exposed either to doxorubicin at 2 µM or to BGNPs at concentrations corresponding to ¼ IC50, ½ IC50, and IC50. After 48 h of treatment, untreated and treated A549 cells were harvested for further analysis. Cells were detached using trypsin–EDTA, collected by centrifugation, and washed twice with ice-cold PBS to remove residual medium, trypsin, and unbound nanoparticles. The resulting cell pellets were resuspended in PBS and stored at −80 °C to preserve molecular integrity. All treatments were performed in triplicate to ensure reproducibility and minimize variability.
Estimation of genomic DNA integrity in A549 cancer cells
The integrity of genomic DNA of non-small A549 lung cancer cells exposed to the Doxorubicin (2 µM/ml) or BGNPs at ¼ IC50, ½ IC50, and IC50 concentrations was assessed using the alkaline comet assay, following the protocols described by Tice et al. (2000) and Langie et al. (2015). Briefly, a suspension of control and Doxorubicin- or BGNPs-treated A549 cells was mixed with 0.5% low-melting agarose at 37 °C and immediately layered onto pre-coated microscope slides containing a basal layer of 1% normal-melting agarose. After solidification at 4 °C, slides were immersed in freshly prepared cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10) supplemented with 1% Triton X-100 and 10% DMSO, and incubated at 4 °C in the dark for 24 h to remove proteins and membranes. Subsequently, slides were transferred to alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 15 min at 4 °C to allow DNA unwinding, followed by electrophoresis at 25 V and 300 mA for 30 min under chilled conditions. After electrophoresis, slides were neutralized with 0.4 M Tris-HCl buffer (pH 7.5), fixed in methanol, air-dried, and stained with ethidium bromide (20 µg/ml) for 10 min in the dark. Fluorescent comets were visualized using fluorescence microscope, and quantitative analysis of DNA damage was carried out using COMETSCORE™ software (TriTek Corp., USA). The extent of DNA fragmentation was expressed as tail length, %DNA in the tail, and tail moment. These parameters were compared between untreated control cells and treated A549 cancer cells to determine the influence of BGNPs genomic stability in A549 cells.
Analysis of intracellular ROS generation in A549 cancer cells
Intracellular ROS production in A549 lung cancer cells after 48 h of exposure to the doxorubicin (2 µM/ml) or BGNPs concentrations (¼ IC50, ½ IC50, and IC50) was measured using the fluorescent probe 2,7-dichlorofluorescin diacetate (2,7-DCFH-DA), following a protocol described by Siddiqui et al. (2010). Untreated and treated A549 cells were harvested, resuspended in PBS at 1 × 10⁶ cells/ml, and incubated with 20 µM 2,7-DCFH-DA in the dark for 30 min at room temperature. The non-fluorescent probe diffused into the cells and was converted intracellularly to fluorescent dichlorofluorescein in the presence of ROS, with fluorescence intensity reflecting ROS levels. Stained A549 cells were mounted on clean slides and observed under an epifluorescence microscope at ×200 magnification. Images were analyzed using Fiji (ImageJ), and ROS levels in BGNPs-treated cells were statistically compared to control cells. The assay was performed in triplicate to ensure reliability.
Assessment of oxidative stress markers
Induction of oxidative stress was assessed by measuring the level of malondialdehyde (MDA), a marker of lipid peroxidation. As described by Ohkawa et al. (1979), MDA reacts with thiobarbituric acid (TBA) under acidic conditions at 95 °C to form a pink MDA–TBA complex. The absorbance of the resulting product was measured at 534 nm using a spectrophotometer, and the MDA level was expressed in mmol/ml. Moreover, the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was estimated using the methods of Nishikimi et al. (1972) and Aebi (1984), respectively. SOD activity was determined based on its inhibition of the phenazine methosulfate-mediated reduction of nitroblue tetrazolium, while CAT activity was measured by the enzyme’s decomposition of hydrogen peroxide (H₂O₂). SOD and CAT activities were expressed in units per milliliter (U/ml).
Estimation of mitochondrial membrane potential in A549 cancer cells
Mitochondrial membrane potential, an indicator of mitochondrial integrity and early apoptosis, was estimated in A549 lung cancer cells following treatment with doxorubicin (2 µM/ml) or BGNPs at various concentrations (¼ IC50, ½ IC50, and IC50) for 48 h. Using a protocol previously reported by Zhang et al. (2011), untreated and treated A549 cells were stained with rhodamine-123, a fluorescent dye that accumulates in active mitochondria. After treatment, cells were harvested, resuspended in PBS, and incubated with rhodamine-123 (10 µg/ml) for 1 h at 37 °C in the dark. Following washes to remove excess dye, cells were spread on clean slides and examined by epifluorescence microscopy at ×200 magnification. Fluorescence intensity, monitoring mitochondrial polarization, was quantified using Fiji (ImageJ). Treated cells showed reduced fluorescence, indicating loss of mitochondrial membrane potential and early apoptosis. Experiments were performed in triplicate, with data reported as mean ± SD.
Analysis of p53, Bax, and Bcl2 gene expression in A549 cancer cells
To evaluate the impact of BGNPs on apoptosis and mitochondrial function, mRNA levels of the pro-apoptotic p53 and Bax genes and anti-apoptotic Bcl2 gene were measured in A549 lung cancer cells after 48 h of treatment with doxorubicin (2 µM/ml) or BGNPs at ¼ IC50, ½ IC50, and IC50 concentrations using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from treated and control cells using the GeneJET RNA Purification Kit, and RNA quality and concentration were assessed by NanoDrop. One microgram of RNA was reverse transcribed to cDNA with the High-Capacity cDNA Reverse Transcription Kit. qRT-PCR was performed using SYBR Green Master Mix and gene-specific primers listed in Table 1 (Suzuki et al. 1999; Lai et al. 2013), with GAPDH as the internal control. Reactions were run in triplicate on a StepOnePlus system. Gene expression changes were calculated using the 2^ − ΔΔCt method and reported as mean ± SD from three independent experiments, revealing BGNPs-induced modulation of apoptotic and mitochondrial gene expression in A549 cancer cells. Table 1. Sequences of the used primers in qRT-PCRGeneStrandPrimer’s sequencesBAXForward5′-CCGCCGTGGACACAGAC-3′Reverse5′-CAGAAAACATGTCAGCTGCCA-3′BCL2Forward5′-TCCGATCAGGAAGGCTAGAGT-3′Reverse5′-TCGGTCTCCTAAAAGCAGGC-3′P53Forward5′-CAGCCAAGTCTGTGACTTGCACGTAC-3′Reverse5′-CTATGTCGAAAAGTGTTTCTGTCATC-3′GAPDHForward5′-GAAGGTGAAGGTCGGAGTCA-3′Reverse5′-GAAGATGGTGATGGGATTTC-3′
Statistical analysis
All data of this study are presented as mean ± standard deviation (SD) and were analyzed using SPSS version 20. One-way analysis of variance (ANOVA) followed by Duncan’s post test was done to compare untreated control and doxorubicin- or BGNPs-treated A549 cells. Regression and correlation analyses were also carried out to assess the relationship between BGNPs tested concentrations and changes in genomic DNA integrity, oxidative stress markers, and gene expression in A549 cancer cells.
Results
Characterization of BGNPs
Characterization results shown in Fig. 1 illustrate the structural and morphological characteristics of BGNPs. XRD analysis displayed a broad, diffuse peak within the 2θ range of 15.48° to 35.11°, indicating an amorphous glass structure with no long-range crystalline order. Such amorphous characteristics are typical of bioactive glass and contribute to enhanced surface reactivity and bioactivity. TEM imaging revealed that the BGNPs were mainly spherical, well-dispersed, and exhibited minimal aggregation. The average particle size was approximately 50 ± 20 nm, confirming their nanoscale dimensions (Fig. 1).Fig. 1. Characterization of BGNPs showing (a) their characteristics XRD profile and (b) their morphological features observed by TEM
BGNPs exhibit strong targeted cytotoxicity toward A549 lung cancer cells
Results of MTT cytotoxicity assay revealed a strong, concentration-dependent decrease in A549 lung cancer cell viability following 48-h exposure to BGNPs at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μg/ml (Fig. 2). The IC50 value for A549 cells was 11.56 μg/ml, indicating potent cytotoxicity of BGNPs toward A549 cancer cells. In contrast, normal HSF cells showed minimal sensitivity to BGNPs under the same conditions, with an IC50 of 1437 μg/ml (Fig. 2). This large difference resulted in a selectivity index (SI) of 124.31, highlighting the high specificity of BGNPs toward A549 lung cancer cells while sparing normal HSF cells. Based on these findings, further molecular analyses were conducted on A549 cells treated with doxorubicin (2 µM/ml) or BGNPs at ¼ IC50, ½ IC50, and IC50 concentrations (2.89, 5.78, and 11.56 µg/ml, respectively) to investigate their therapeutic potential.Fig. 2. Viability of normal HSF cells and A549 lung cancer cells after 48-h exposure to varying concentrations of BGNPs (0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 µg/ml)
BGNPs significantly disrupt genomic stability in A549 lung cancer cells
Results of alkaline single-cell comet assay revealed substantial genomic DNA damage in A549 cancer cells treated with BGNPs at ¼ IC50 (2.89 µg/ml), ½ IC50 (5.78 µg/ml), and IC50 (11.56 µg/ml) for 48 h in a concentration-dependent manner. All BGNPs-treated cells showed statistically significant increases (p < 0.001) in DNA damage markers, including tail length, % DNA in tail, and tail moment, compared to both untreated and doxorubicin-treated control A549 cells (Table 2). Representative comet images shown in Fig. 3 visually supported these findings. While control cells displayed compact nuclei indicative of intact DNA, BGNPs-treated cells exhibited extensive DNA migration, forming distinct comet tails. Notably, DNA fragmentation was more pronounced in BGNPs-treated cells than in those treated with doxorubicin. Regression and correlation analyses confirmed a strong concentration-dependent effect. Significant positive correlations were observed between BGNPs tested concentration and tail length (r = + 0.99), % DNA in tail (r = + 0.97), and tail moment (r = + 0.99), indicating that genomic instability increased consistently with rising BGNPs concentrations (Fig. 4). Table 2. Incidence of genomic DNA damage in untreated control and A549 lung cancer cells treated with doxorubicin (2 µM) or varying concentrations of BGNPs (2.89, 5.78, and 11.56 µg/ml) for 48 hCellsTreatmentConcentrationTail length (px)%DNA in tailTail momentA549 lung cancer cellsUntreated cells0.003.39 ± 0.45^a^11.71 ± 1.73^a^0.40 ± 0.07 ^a^Doxorubicin2 µM/ml10.87 ± 1.35^b^21.04 ± 1.35 ^b^2.24 ± 0.10 ^b^Bioactive glass nanoparticles1/4 IC50 (2.89 µg/ml)15.22 ± 0.93^c^30.11 ± 0.50 ^c^4.51 ± 0.15 ^c^1/2 IC50 (5.78 µg/ml)22.19 ± 1.07 ^d^45.58 ± 1.54 ^d^10.90 ± 1.79 ^d^IC50 (11.56 µg/ml)35.91 ± 1.53 ^e^60.40 ± 2.33 ^e^21.45 ± 1.49 ^e^ANOVAF** = 358.17; p < 0.001F = 437.39; p < 0.001F = 200.40; p < 0.001Results are expressed as mean ± SD. Results were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s test. Means with different superscript letters indicate statistical significant difference among the compared untreated control and treated A549 lung cancer cells at p < **0.001Fig. 3Representative examples for the scored comet nuclei with intact DNA in untreated A549 lung cancer cells and those with damaged DNA in A549 cells treated with doxorubicin (2 µM) or varying concentrations of BGNPs (2.89, 5.78, and 11.56 µg/ml) for 48 hFig. 4Regression line and correlation coefficient between BGNPs tested concentrations (2.89, 5.78, and 11.56 µg/ml) and DNA damage parameters: tail length, %DNA in tail and tail moment
BGNPs significantly elevate ROS generation in A549 cancer cells
As displayed in Fig. 5 and 6, BGNPs induced a marked, concentration-dependent increase in intracellular ROS levels in A549 lung cancer cells after 48-h exposure to ¼ IC50 (2.89 µg/ml), ½ IC50 (5.78 µg/ml), and IC50 (11.56 µg/ml) concentrations. This oxidative stress was confirmed by a highly significant rise (p < 0.001) in fluorescence intensity of the ROS-sensitive dye 2,7-DCFH-DA, which fluoresces upon oxidation by ROS (Fig. 5). Fluorescence microscopy revealed stronger green signals in BGNPs-treated cells compared to untreated and doxorubicin-treated control A549 cells, indicating elevated cytoplasmic ROS accumulation. Notably, ROS levels in BGNPs-treated A549 cells surpassed those observed with doxorubicin, highlighting the potent oxidative effect of BGNPs (Fig. 6).Fig. 5. Measurement of ROS production in A549 lung cancer cells treated with doxorubicin (2 µM) or BGNPs (2.89, 5.78, 11.56 µg/ml) for 48 h, with untreated cells as control. Data are expressed as mean ± SD and analyzed using one-way ANOVA followed by Duncan’s test. Different letters indicate statistically significant differences at p < 0.001Fig. 6ROS generation levels in A549 lung cancer cells following 48 h treatment with doxorubicin (2 µM) or BGNPs (2.89, 5.78, 11.56 µg/ml) relative to untreated control cells
BGNPs induce oxidative stress in A549 cancer cells
In line with ROS generation results, biochemical assays confirmed a significant, concentration-dependent induction of oxidative stress in A549 lung cancer cells following 48-h exposure to BGNPs at various concentrations. As reported in Table 3, exposure to BGNPs at concentrations of 2.89, 5.78, and 11.56 µg/ml for 48 h led to a marked, concentration-dependent increase (p < 0.001) in MDA level, a key indicator of lipid peroxidation, compared to both untreated and doxorubicin-treated cells, exceeding those induced by doxorubicin. Concurrently, the activities of the antioxidant enzymes CAT and SOD significantly declined (p < 0.001) in a concentration-dependent manner, with reductions greater than those observed in doxorubicin-treated cells (Table 3). Regression analysis further validated these observations, revealing a strong positive correlation between BGNPs concentration and MDA level (r = + 0.99), and strong negative correlations with CAT (r = –0.99) and SOD (r = –0.97) activities, as depicted in Fig. 7. Table 3. Level of MDA and activity of antioxidant CAT and SOD enzymes in untreated control and A549 lung cancer cells treated with doxorubicin (2 µM) or varying concentrations of BGNPs (2.89, 5.78, and 11.56 µg/ml) for 48 hCellsTreatmentConcentrationMDA level (mmol/ml)CAT activity (U/L)SOD activity (U/ml)A549 lung cancer cellsControl cells0.005.19 ± 0.03^a^784.07 ± 4.57^e^322.61 ± 9.07 ^e^Doxorubicin2 µM/ml22.58 ± 1.32^c^349.89 ± 6.51 ^b^141.38 ± 7.54 ^b^BGNPS1/4 IC50 (2.89 µg/ml)11.26 ± 1.16^b^662.87 ± 4.13 ^d^243.03 ± 6.83 ^d^1/2 IC50 (5.78 µg/ml)21.20 ± 2.01 ^c^562.75 ± 3.37 ^c^177.98 ± 2.99 ^c^IC50 (11.56 µg/ml)35.08 ± 3.83 ^d^329.20 ± 4.09 ^a^119.96 ± 4.91^a^ANOVAF** = 90.55; p < 0.001F = 5380.32; p < 0.001F = 466.45; p** < 0.001Results are expressed as mean ± SD. Results were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s test. Means with different superscript letters indicate statistical significant difference among the compared untreated control and treated A549 lung cancer cells at p < **0.001Fig. 7Regression analysis showing correlations between BGNPs concentrations (2.89, 5.78, 11.56 µg/ml) and oxidative stress markers, including MDA level and SOD/CAT antioxidant enzyme activities
BGNPs induce mitochondrial membrane deplorization in A549 cancer cells
As depicted in Fig. 8 and 9, exposure of A549 lung cancer cells to BGNPs for 48 h at ¼ IC50 (2.89 µg/ml), ½ IC50 (5.78 µg/ml), and IC50 (11.56 µg/ml) concentrations resulted in a significant, concentration-dependent loss of mitochondrial membrane potential. This was evidenced by a marked reduction in rhodamine-123 fluorescence intensity (p < 0.001) compared to both untreated and doxorubicin-treated control cells (Fig. 8). The fluorescence decrease was more pronounced in BGNPs-treated A549 cells than in those exposed to doxorubicin, indicating greater mitochondrial depolarization. These findings highlight the strong disruptive effect of BGNPs on mitochondrial integrity in A549 cells (Fig. 9).Fig. 8. Assessment of mitochondrial membrane potential in A549 cells treated with doxorubicin (2 µM) or BGNPs (2.89, 5.78, 11.56 µg/ml) for 48 h. Data are expressed as mean ± SD and analyzed by one-way ANOVA with Duncan’s test. Different letters denote significant differences at p < 0.001Fig. 9Mitochondrial membrane potential integrity in untreated and treated A549 cells (doxorubicin 2 µM or BGNPs 2.89–11.56 µg/ml) after 48 h, highlighting treatment-dependent variation
BGNPs significantly dysregulate p53, Bax, and Bcl2 expression in A549 cancer cells
The findings of qRT-PCR) analysis, shown in Table 4, revealed significant, concentration-dependent dysregulation in the expression of key apoptotic (p53 and Bax) and anti-apoptotic (Bcl-2) genes in A549 lung cancer cells following 48-h treatment with BGNPs at concentrations of 2.89, 5.78, and 11.56 µg/ml. The expression level of the pro-apoptotic p53 and Bax genes was significantly upregulated (p < 0.001) in a concentration-dependent manner compared to both doxorubicin-treated and untreated control cells, suggesting activation of a p53-mediated apoptotic pathway in response to BGNPs exposure. Conversely, the expression of the anti-apoptotic Bcl-2 gene was significantly downregulated (p < 0.001) in BGNPs-treated cells compared to doxorubicin-treated and untreated control cells (Table 4), indicating suppression of anti-apoptotic signaling mechanisms. These transcriptional changes were further supported by regression analysis, which showed strong positive correlations between BGNPs tested concentrations and the expression of p53 (r = + 0.95) and Bax (r = + 0.90), along with a strong negative correlation with Bcl-2 gene expression (r = –0.89), as illustrated in Fig. 10. Table 4. Expression level of apoptotic (p53 and Bax) and anti-apoptotic Bcl2 genes in in untreated control and A549 lung cancer cells treated with doxorubicin (2 µM) or varying concentrations of BGNPs (2.89, 5.78, and 11.56 µg/ml) for 48 hTreatmentConcentrationFold change in the expression level ofp53 geneBax geneBcl2 geneUntreated cells0.001.00 ± 0.00^a^1.00 ± 0.00^a^1.00 ± 0.00 ^a^Doxorubicin2µM/ml2.59 ± 0.04^b^3.75 ± 0.01 ^b^0.67 ± 0.02 ^b^Y_2_O_3_NPs¼ IC50 (2.89 µg/ml)4.39 ± 0.01^c^5.25 ± 0.04 ^c^0.42 ± 0.03 ^b^½ IC50 (5.78 µg/ml)7.55 ± 0.04 ^d^9.23 ± 0.04 ^d^0.28 ± 0.02 ^c^IC50 (11.56 µg/ml)9.32 ± 0.03 ^e^12.20 ± 0.03 ^e^0.08 ± 0.01 ^d^ANOVAF** = 45528.73; p < 0.001F = 72364.37; p < 0.001F = 893.69; p < 0.001Results are expressed as mean ± SD. Results were analyzed using One Way Analysis of Variance (ANOVA) followed by Duncan’s test. Means with different superscript letters indicate statistical significant difference among the compared untreated control and treated A549 lung cancer cells at p < **0.001Fig. 10Regression lines and correlation coefficient between BGNPs tested concentrations (2.89, 5.78, and 11.56 µg/ml) and expression level of p53, Bax, and Bcl2 genes
Discussion
Non-small cell lung cancer (NSCLC), which accounts for the majority of lung cancer cases, continues to face significant therapeutic challenges. Conventional chemotherapy, the cornerstone of NSCLC management, is often limited by nonspecific cytotoxicity, the emergence of multidrug resistance, severe systemic side effects, and limited efficacy in improving long-term survival (Molina et al. 2008; Siegel et al. 2023). These drawbacks underscore the urgent need for novel and more selective therapeutic strategies. Despite the growing interest in nanomedicine for cancer therapy, there remains a notable gap in the literature regarding the therapeutic potential of BGNPs in lung cancer. While bioactive glasses have been extensively studied for their applications in bone regeneration and tissue engineering due to their excellent biocompatibility, bioactivity, and biodegradability (Hench 2006; Hussein et al. 2025), their cytotoxic and anticancer properties remain poorly explored, especially in the context of lung malignancies. Consequently, investigating the selective cytotoxicity of BGNPs, their underlying mechanisms of action, and molecular effects in the A549 NSCLC model in this study could unveil promising avenues for more targeted and safer cancer therapies.
In the present study, BGNPs demonstrated a potent, selective cytotoxic effect against A549 lung cancer cells, while sparing normal human HSF cells, as indicated by a very high selectivity index of 124.31, consistent with the findings of Fellenberg et al. (2022) and Deliormanlı et al. (2024), which reported strong cytotoxic effects of macroparticulate bioactive glass on osteosarcoma (SaOS-2) and giant cell tumor cells, alongside minimal toxicity to normal bone-derived stromal cells, further supporting the potential of bioactive glass-based materials in selective cancer therapy. This selective toxicity is a critical advantage in cancer treatment, as conventional chemotherapeutic agents like doxorubicin often damage healthy cells, leading to severe side effects.
As illustrated in Fig. 11, the mechanistic investigations demonstrated that BGNPs induced cytotoxicity in A549 lung cancer cells through multiple interconnected pathways. Central to these effects was the excessive generation of ROS, which triggered oxidative stress, subsequently leading to substantial disruption of genomic DNA integrity, impairment of mitochondrial function, and significant transcriptional alterations in apoptosis-related genes. Specifically, BGNPs treatment resulted in the upregulation of pro-apoptotic p53 and Bax genes, alongside the downregulation of the anti-apoptotic Bcl2 gene, collectively promoting A549 cell death. Marked disruption of genomic DNA integrity was clearly demonstrated in BGNPs-treated A549 lung cancer cells through the significant concentration-dependent increases in DNA damage markers, tail length, %DNA in tail, and tail moment, assessed by the alkaline comet assay. Notably, the extent of DNA damage induced by BGNPs surpassed that observed with doxorubicin, highlighting their potent genotoxic potential. These findings are consistent with earlier studies showing that nanomaterials can induce DNA strand breaks and genotoxic stress primarily through ROS-mediated mechanisms (Mesárošová et al. 2014; Mohamed et al. 2025a, b, c; Mohamed et al. 2025a).Fig. 11. Schematic illustration of the potent, targeted cytotoxic effects of BGNPs on A549 lung cancer cells and the proposed underlying mechanisms
Consistent with the observed genotoxicity, a marked concentration-dependent elevation in intracellular ROS level was detected in BGNPs-treated A549 cells, exceeding levels induced by doxorubicin. This excessive ROS generation is likely a central contributor to oxidative DNA damage, lipid peroxidation, and mitochondrial dysfunction. ROS are known to play a dual role in cancer biology; while regulated ROS levels can promote tumor progression, excessive and unbalanced ROS accumulation induces oxidative stress, ultimately leading to apoptotic cell death (Trachootham et al*.*2009). Our biochemical analyses further validated the induction of oxidative stress in BGNPs-treated A549 lung cancer cells, as evidenced by a concentration-dependent, significant elevation in MDA level, a hallmark of lipid peroxidation, alongside a marked depletion in the antioxidant enzymes CAT and SOD. These findings are in line with earlier studies demonstrating nanoparticle-induced oxidative stress in various cancer models (Siddiqi et al. 2018; Subramaniam et al. 2024; Mohamed et al. 2025b).
In line with our findings, growing evidence indicates that nanoparticles can induce oxidative stress and DNA damage, processes central to carcinogenesis and therapeutic response. For instance, engineered nanomaterials have been shown to exert genotoxic effects by disrupting DNA integrity, as highlighted in the study of Singh and colleagues (2009). Similarly, nanoparticle-induced DNA damage has been reported to mimic signaling cascades activated during irradiation-induced carcinogenesis, further supporting the mechanistic relevance of ROS generation and DNA strand breaks in cancer biology (Mroz et al. 2008; Singh et al. 2009). More recently, Feola et al. (2024) demonstrated that polystyrene nanoparticles induce DNA damage and apoptosis in HeLa cells, further confirming the reproducibility of these effects across different tumor types. Moreover, Nebbioso et al. (2011) reported that the epigenetic inhibitor UVI5008 enhances ROS production and activates the death receptor pathway, thereby underscoring the close interplay between oxidative stress and programmed cell death in oncological models. Collectively, these studies strengthen our results and suggest that BGNPs may mediate their anticancer activity through conserved mechanisms involving oxidative stress, DNA damage, and apoptosis, supporting their potential as versatile therapeutic agents across different malignancies.
Furthermore, BGNPs caused a substantial collapse in mitochondrial membrane potential in A549 cancer cells, as indicated by a significant concentration-dependent reduction in rhodamine-123 fluorescence intensity after 48 h of A549 cancer cell treatment with BGNPs at 2.89, 5.78, and 11.56 µg/ml, surpassing the reduction induced by doxorubicin treatment. The loss of mitochondrial integrity is a hallmark of intrinsic apoptosis and serves as a central mediator of ROS-driven cytotoxicity (Wang and Youle 2009). The mitochondrial dysfunction observed here in BGNPs-treated A549 likely results from ROS overload, triggering the release of apoptogenic factors and activation of downstream apoptotic pathways (Mohamed et al. 2025a, b, c).
To further validate apoptosis induction, we assessed the expression of key apoptotic and anti-apoptotic genes. qRT-PCR analysis demonstrated concurrent significant upregulation of pro-apoptotic p53 and Bax genes and pronounced downregulation of the anti-apoptotic Bcl-2 gene in a concentration-dependent manner. These molecular alterations strongly support the hypothesis that BGNPs trigger apoptosis through a p53-mediated mitochondrial pathway, consistent with prior research indicating nanoparticle-induced apoptosis through modulation of p53 and Bax expression (Mohamed et al. 2022, 2025a; Szewczyk-Roszczenko and Barlev 2023). Together, the observed gene expression changes, coupled with oxidative and mitochondrial damage, illustrate a multi-faceted mechanism of BGNP-induced cytotoxicity in A549 lung cancer cells.
Despite the compelling evidence for the strong and selective cytotoxicity of BGNPs against A549 lung cancer cells, several limitations should be acknowledged. First, our experiments were conducted exclusively on A549 NSCLC cells, which, although widely used as a model for lung adenocarcinoma, may not fully capture the heterogeneity of NSCLC subtypes; future studies should assess BGNPs’ activity across multiple NSCLC cell lines to confirm the generalizability of these effects. Second, while cytotoxicity and mitochondrial dysfunction were evaluated at specific concentrations and time points, a more comprehensive dose-time analysis is necessary to fully characterize pharmacodynamics and optimize therapeutic windows. Furthermore, these in vitro results, although mechanistically informative, do not account for the complexity of the tumor microenvironment, including interactions with stromal cells, immune components, and extracellular matrix factors, which may influence BGNPs’ efficacy and ROS-mediated responses; in vivo validation is therefore required to determine therapeutic efficacy and biosafety. Third, the selectivity of BGNPs between cancerous and normal lung epithelial cells was not examined, an essential consideration for potential clinical translation. Finally, the potential for BGNPs to act synergistically with existing therapies remains unexplored and warrants further investigation to enhance treatment outcomes and minimize adverse effects. Collectively, these considerations underscore the need for further research using diverse NSCLC models, physiologically relevant systems, and combination treatment strategies to validate and expand the translational potential of BGNPs in lung cancer therapy.
Although our comparison of BGNPs with doxorubicin provides a useful reference, it is limited by mechanistic differences: BGNPs primarily act through oxidative stress, mitochondrial dysfunction, and apoptosis, whereas doxorubicin functions via topoisomerase II inhibition and DNA intercalation. Pharmacokinetic and bio-distribution differences further complicate direct comparisons. Nevertheless, these distinct mechanisms suggest the potential for therapeutic synergy when BGNPs are combined with conventional chemotherapies such as doxorubicin. Indeed, previous studies have demonstrated that nanomaterials can sensitize cancer cells to chemotherapy, reduce drug resistance, and allow dose reduction, thereby minimizing systemic toxicity (Li et al. 2021; Alrohaim et al. 2025; Kalındemirtaş et al. 2025). Exploring such combination strategies could enhance therapeutic efficacy and broaden the clinical applicability of BGNPs, representing a promising avenue for future research.
Conclusion
Collectively, the data presented demonstrate that BGNPs exert potent and highly selective cytotoxic effects on A549 NSCLC cells, as evidenced by a high selectivity index of 124.31. This cytotoxicity is mediated through multiple mechanisms, including oxidative stress, DNA damage, mitochondrial dysfunction, and modulation of apoptotic gene expression. In contrast, BGNPs exhibited no observable toxicity in normal HSF cells under the same treatment conditions. These findings address a critical gap in our understanding of BGNPs’ anticancer potential in lung malignancies and highlight their promise as candidates for further preclinical development as targeted therapeutics for NSCLC. Notably, their multi-mechanistic mode of action positions BGNPs as attractive agents for use in combination or standalone therapies, with the potential to overcome resistance associated with conventional single-target chemotherapeutics.