NADPH oxidase-produced superoxide mediated a 50-Hz magnetic field-induced epidermal growth factor receptor clustering

Purpose: A 50-Hz magnetic field (MF) was found to induce epidermal growth factor receptor (EGFR) clustering in our previous study. The aim of this work was to investigate the molecular mechanisms that mediated MF-induced EGFR clustering.

Materials and methods: Human amniotic epithelial (FL) cells were exposed to a 50-Hz MF. Total reactive oxygen species (ROS), cytoplasmic and mitochondrial superoxide production were detected by DCFH-DA, DHE and MitoSOX, respectively. EGFR clustering was analyzed using confocal microscopy after indirect immunofluorescence staining.

Results: Results showed that exposing FL cells to MF at intensity higher than 0.2?mT for 15?min enhanced total ROS production. Additionally, enhanced total ROS and cytoplasmic superoxide production were observed after exposing cells to MF at 0.4?mT for 5, 15, or 30?min, while mitochondrial superoxide production for 15 or 30?min. Pretreatment with Nox inhibitor, DPI, effectively inhibited MF-induced cytoplasmic superoxide production and subsequent EGFR clustering while mitochondrial superoxide production was not affected.

Conclusions: Nox-produced superoxide mediated a 50-Hz magnetic field-induced EGFR clustering.     

Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation

Purpose: Exposure to extremely low frequency electromagnetic fields (ELF-EMFs) could elicit biological effects including carcinogenesis. However, the detailed mechanisms by which these ELF-EMFs interact with biological system are currently unclear. Previously, we found that a 50-Hz magnetic field (MF) exposure could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell membranes. In the present experiment, the possible roles of reactive oxygen species (ROS) in MF-induced EGFR clustering were investigated.

Materials and methods: Human amnion epithelial (FL) cells were exposed to a 50-Hz MF with or without N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). EGFR clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining. The intracellular ROS level and acid sphingomyelinase (ASMase) activity were detected using an ROS assay kit and an Amplex^® Red Sphingomyelinase Assay Kit, respectively.

Results: Results showed that exposure of FL cells to a 50-Hz MF at 0.4?mT for 15?min significantly enhanced the ROS level, induced EGFR clustering and increased ASMase activity. However, pretreatment with NAC or PDTC, the scavenger of ROS, not only counteracted the effects of a 50-Hz MF on ROS level and AMS activity, but also inhibited the EGFR clustering induced by MF exposure.

Conclusions: The present and previous data suggest that ROS mediates the MF-induced EGFR clustering via ASMase activation.     

50-Hz magnetic field impairs the expression of iron-related genes in the in vitro SOD1G93A model of amyotrophic lateral sclerosis

Abstract

Purpose: We characterized the response to the extremely low frequency magnetic field (ELF-MF) in an in vitro model of familial Amyotrophic Lateral Sclerosis (fALS), carrying two mutant variants of the superoxide dismutase 1 (SOD1) gene.

Materials and methods: SH-SY5Y human neuroblastoma cells, stably over-expressing the wild type, the G93A or the H46R mutant SOD1 cDNA, were exposed to either the ELF-MF (50?Hz, 1?mT) or the sham control field, up to 72?h. Analysis of (i) viability, proliferation and apoptosis, (ii) reactive oxygen species generation, and (iii) assessment of the iron metabolism, were carried out in all clones in response to the MF exposure.

Results: We report that 50-Hz MF exposure induces: (i) no change in proliferation and viability; (ii) no modulation of the intracellular superoxide and H₂O₂ levels; (iii) a significant deregulation in the expression of iron-related genes IRP1, MFRN1 and TfR1, this evidence being exclusive for the SOD1^G93A clone and associated with a slight (p?=?.0512) difference in the total iron content.

Conclusions: 50-Hz MF affects iron homeostasis in the in vitro SOD1^G93A ALS model.     

Exposure to a 50-Hz magnetic field induced ceramide generation in cultured cells

Purpose To investigate the effects of a 50-Hz magnetic field (MF) exposure on ceramide metabolism, as well as the cascade downstream signaling pathways in human amniotic (FL) cells.

Materials and methods FL cells were exposed to MF at 0.4?mT for different durations (from 5–60?min). The ceramides levels were analyzed with high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The activity of cathepsin D was assayed using a fluorometric assay kit, and the activity of protein phosphatase 2A (PP2A) was examined by Western blotting. After exposing to MF at 0.4?mT for 60?min with sequential culture for different durations (0, 3, 6, 12 or 36?h), the rate of cell apoptosis was assessed by flow cytometry.

Results Exposing cells to MF at 0.4?mT for different durations caused a significant increase in ceramide production via de novo synthesis and hydrolysis of sphingomyelin (SM), and the effect was different according to the exposure time. However, no significant change in cell apoptosis was detected after MF exposure for 60?min with sequentially culturing for up to 36?h. In addition, increase in ceramide did not activate its downstream signal molecules, cathepsin D and PP2A, which are usually closely related to apoptosis of cells.

Conclusions Exposure to a 50-Hz MF could raise ceramide levels but had no significant effect on apoptosis in cultured cells.     

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells

Abstract

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy.

Material and methods: Cells were exposed to EMFs (25, 50 and 100?Hz; 8 and 10?mT). In the co-treatment, cells were first exposed to EMFs (50?Hz/10?mT) for 30?min and then to ionizing radiation (IR) (2?Gy) 4?h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay.

Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4?h after exposure to 50?Hz/10?mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24?h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells.

Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.     

Effects of single- and hybrid-frequency extremely low-frequency electromagnetic field stimulations on long-term potentiation in the hippocampal Schaffer collateral pathway

Abstract

Purpose: To study the different effects of single- and hybrid-frequency magnetic fields on long-term potentiation (LTP) in synaptic plasticity.

Materials and methods: Based on the online electromagnetic field stimulation system and field excitatory postsynaptic potentials (fEPSPs) recording system, we applied four different single- and hybrid-frequency magnetic fields with an intensity of 1?mT to the Schaffer collateral (CA1) pathway of rat hippocampal slices in vitro.

Results: The amplitude of fEPSPs decreased significantly under both single- and hybrid-frequency magnetic stimulation. Lower single-frequency magnetic stimulation on LTP had a greater regulating effect, while the regulating effect among four different hybrid-frequency extremely low-frequency electromagnetic fields (ELF-EMFs) stimulations on LTP showed no significant differences.

Conclusion: Single-frequency magnetic stimulation produces more significant regulatory effects, and the lower the frequency, the more significant the regulatory effect. The effect of hybrid-frequency magnetic stimulation in each group was similar, and there was no significant difference between each group. The 15-Hz single-frequency magnetic stimulation group showed the most significant regulatory effect, but once it was mixed with other higher frequency magnetic stimulation, its regulation effect was significantly weakened.     

Infrared spectroscopic demonstration of magnetic orientation in SH-SY5Y neuronal-like cells induced by static or 50 Hz magnetic fields

Abstract

Purpose: To study the response of neuronal-like cells to an applied static or low-frequency magnetic field.

Materials and methods: Fourier Transform Infrared (FTIR) Spectroscopy was used to investigate the overall behavior of SH-SY5Y neuronal-like cells exposed to a static or 50?Hz magnetic fields at intensities up to 1?mT generated by two Helmholtz coils driven by direct current or alternate current voltage. The Helmholtz coils were placed into an incubator in a 5% CO₂/95% air humidified at 37?°C.

Results: A significant increase in intensity of the Amide I band and of CH₂ stretching vibrations in neuronal-like FTIR spectra appeared after 6?h exposure to static or 50?Hz magnetic fields.

Conclusions: These findings can be easily explained assuming that proteins α-helices and lipids contents in cellular membrane aligned toward the direction of the applied magnetic field after exposure, inducing an increase of ions flux across cellular membrane channels after exposure to a magnetic field, changing cellular functions.     

Enhancement of biological effectiveness of carbon-ion beams by applying a longitudinal magnetic field

Abstract

Purpose: A magnetic field longitudinal to an ion beam will potentially affect the biological effectiveness of the radiation. The purpose of this study is to experimentally verify the significance of such effects.

Methods and materials: Human cancer and normal cell lines were exposed to low (12?keV/μm) and high (50?keV/μm) linear energy transfer (LET) carbon-ion beams under the longitudinal magnetic fields of B_// = 0, 0.1, 0.2, 0.3, or 0.6 T generated by a solenoid magnet. The effects of the magnetic fields on the biological effectiveness were evaluated by clonogenic cell survival. Doses that would result in a survival fraction of 10% (D₁₀s) were determined for each cell line and magnetic field.

Results: For cancer cells exposed to the low (high)-LET beams, D₁₀ decreased from 5.2 (3.1) Gy at 0 T to 4.3 (2.4) Gy at 0.1 T, while no further decrease in D₁₀ was observed for higher magnetic fields. For normal cells, decreases in D₁₀ of comparable magnitudes were observed by applying the magnetic fields.

Conclusions: Significant decreases in D₁₀, i.e. significant enhancements of the biological effectiveness, were observed in both cancer and normal cells by applying longitudinal magnetic fields of B_// ≥?0.1 T. These effects were enhanced with LET. Further studies are required to figure out the mechanism underlying the observed results.     

Extremely low frequency magnetic field (50 Hz, 0.5 mT) modifies fitness components and locomotor activity of Drosophila subobscura

Abstract

Purpose: Extremely low frequency (ELF) magnetic fields are essential ecological factors which may induce changes in many organisms. The aim of this study was to examine the effects in Drosophila subobscura exposed for 48 h to ELF magnetic field (50 Hz, 0.5 mT) at different developmental stages.

Materials and methods: Egg-first instar larvae developmental stage of D. subobscura isofemale lines was exposed to ELF magnetic field, and fitness components (developmental time, developmental dynamics, viability and sex ratio) and locomotor activity of three-day-old males and females were monitored. Also, just eclosed D. subobscura isofemale adults were exposed to ELF magnetic field and their locomotor activity was monitored just after.

Results: ELF magnetic field shortens developmental time, increases viability and does not affect sex ratio of D. subobscura. No matter which developmental stage is exposed, ELF magnetic field significantly decreases locomotor activity of adult flies, but after exposure of just eclosed adults observed change lasts longer.

Conclusions: Applied ELF magnetic field modifies fitness components and locomotor activity of D. subobscura. Observed effects can be attributed to the influence of magnetic field on different stages of development where the hormonal and nervous systems play important role in the control of examined parameters.     

Different responses of Drosophila subobscura isofemale lines to extremely low frequency magnetic field (50 Hz, 0.5 mT): fitness components and locomotor activity

Purpose: Extremely low frequency (ELF) magnetic fields as essential ecological factors may induce specific responses in genetically different lines. The object of this study was to investigate the impact of the ELF magnetic field on fitness components and locomotor activity of five Drosophila subobscura isofemale (IF) lines.

Materials and methods: Each D. subobscura IF line, arbitrarily named: B16/1, B24/4, B39/1, B57/2 and B69/5, was maintained in five full-sib inbreeding generations. Their genetic structures were defined based on the mitochondrial DNA variability. Egg-first instar larvae and 1-day-old flies were exposed to an ELF magnetic field (50?Hz, 0.5?mT, 48?h) and thereafter, fitness components and locomotor activity of males and females in an open field test were observed for each selected IF line, respectively.

Results: Exposure of egg-first instar larvae to an ELF magnetic field shortened developmental time, and did not affect the viability and sex ratio of D. subobscura IF lines. Exposure of 1-day-old males and females IF lines B16/1 and B24/4 to an ELF magnetic field significantly decreased their locomotor activity and this effect lasted longer in females than males.

Conclusions: These results indicate various responses of D. subobscura IF lines to the applied ELF magnetic field depending on their genetic background.     

Exposure to a 50-Hz magnetic field induced mitochondrial permeability transition through the ROS/GSK-3β signaling pathway

Purpose To investigate the biological effects of a 50-Hz magnetic field (MF) on mitochondrial permeability.

Materials and methods Human amniotic epithelial cells were exposed to MF (50?Hz, 0.4?mT) for different durations. Mitochondrial permeability, mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt-c) release and the related mechanisms were explored.

Results Exposure to the MF at 0.4?mT for 60?min transiently induced mitochondrial permeability transition (MPT) and Cyt-c release, although there was no significant effect on mitochondrial membrane potential (ΔΨm). Other than decreasing the total Bcl-2 associated X protein (Bax) level, MF exposure did not significantly affect the levels of Bax and B-cell lymphoma-2 (Bcl-2) in mitochondria. In addition, cells exposed to the MF showed increased intracellular reactive oxidative species (ROS) levels and glycogen synthase kinase-3β (GSK-3β) dephosphorylation at 9 serine residue (Ser⁹). Moreover, the MF-induced MPT was attenuated by ROS scavenger (N-acetyl-L-cysteine, NAC) or GSK-3β inhibitor, and NAC pretreatment prevented GSK-3β dephosphorylation (Ser⁹) caused by MF exposure.

Conclusion MPT induced by MF exposure was mediated through the ROS/GSK-3β signaling pathway.     

Exposure to radiation from single or combined radio frequencies provokes macrophage dysfunction in the RAW 264.7 cell line

Purpose: The aim of this study was to determine whether exposure to radiation from single or multiple radio-frequency (RF) signals at 900 and 2450?MHz would induce effects in the RAW 264.7 cell line.

Materials and methods: Cell cultures were exposed to single or combined RF for 4, 24, 48, or 72?h in a GTEM electromagnetic test chamber. At the end of the radiation exposure time, viability and cell growth were analyzed by flow cytometry, nitric oxide (NO) production was measured by colorimetry, the expression of HSP70 and TNF-α was ascertained by qPCR, and the phagocytic activity was observed by microscopy.

Results: NO production increased after 48?h exposure at 2450?MHz, compared with controls. The group subjected to the combined interaction of two RFs showed an increase of HSP70 after 48?h exposure and a significant increase of NO and TNF-α after 72?h. The phagocytic activity of macrophages decreased in all groups as exposure time increased.

Conclusions: Our results indicated a decrease in phagocytic activity and an increase in inflammatory, cytoprotective, and cytotoxic responses in macrophages after continuous and combined exposure of multiple RF signals. Multiple RF interact in everyday life, the immune response in humans is unknown.     

Modulation of rat synaptosomal ATPases and acetylcholinesterase activities induced by chronic exposure to the static magnetic field

Purpose: It is considered that exposure to static magnetic fields (SMF) may have both detrimental and therapeutic effect, but the mechanism of SMF influence on the living organisms is not well understood. Since the adenosine triphosphatases (ATPases) and acetylcholinesterase (AChE) are involved in both physiological and pathological processes, the modulation of Na⁺/K⁺-ATPase, ecto-ATPases and AChE activities, as well as oxidative stress responses were followed in synaptosomes isolated from rats after chronic exposure toward differently oriented SMF.

Material and methods: Wistar albino rats were randomly divided into three experimental groups (six animals per group): Up and Down group - exposed to upward and downward oriented SMF, respectively, and Control group. After 50 days, the rats were sacrificed, and synaptosomes were isolated from the whole rat brain and used for testing the enzyme activities and oxidative stress parameters.

Results: Chronic exposure to 1?mT SMF significantly increased ATPases, AChE activities, and malondialdehyde (MDA) level in both exposed groups, compared to control values. The significant decrease in synaptosomal catalase activity (1.48?±?0.17?U/mg protein) induced by exposure to the downward oriented field, compared to those obtained for Control group (2.60?±?0.29?U/mg protein), and Up group (2.72?±?0.21?U/mg protein).

Conclusions: It could be concluded that chronic exposure to differently oriented SMF increases ATPases and AChE activities in rat synaptosomes. Since brain ATPases and AChE have important roles in the pathogenesis of several neurological diseases, SMF influence on the activity of these enzymes may have potential therapeutic importance     

Combination of static magnetic field and cisplatin in order to reduce drug resistance in cancer cell lines

Abstract

Purpose: In this study, the effects of different intensities of Static Magnetic Fields (SMFs) (10, 15 and 25?mT) and different concentrations of cisplatin drug were investigated on the viability percent and IC₅₀ of the A2780 and A2780-CP cell lines at 24, 48 and 96?h to show useful potential of SMF as a physical agent to enhance the effectiveness of common therapeutic approaches and decrease of drug resistance to cisplatin anticancer drug.

Materials and methods: Magnetic field exposure was performed using a locally designed generator. The cell viability percent, IC₅₀ and cisplatin uptake in treated cells were evaluated by MTT assay and inductively coupled plasma (ICP), respectively.

Results: Increasing of concentration and time of cisplatin drug showed a noticeable decrease in viability percent in sensitive and resistant cell lines compared with control group. These decreases were more significant in resistant cells compared with sensitive cells. The obtained IC₅₀ values for resistant were greater than the values obtained for A2780 cells. ICP analysis demonstrated an increased uptake of cisplatin after treatment for 48 and 96?h relative to untreated groups in both resistant and sensitive cells.

Conclusion: Results showed that A2780 cells were more sensitive to cisplatin than A2780-CP. Studies have shown that SMF can increase the effect of cisplatin on cell viability percent and decrease the resistance of A2780-CP cells by producing large, verruca shaped structures at the surface of the cell membrane.     

The influence of static magnetic field (50 mT) on development and motor behaviour of Tenebrio (Insecta,Coleoptera)

Abstract

Purpose: There is considerable concern about potential effects associated with exposure to magnetic fields on organisms. Therefore, duration of pupa-adult development and motor behaviour of adults were analyzed in Tenebrio obscursus and T. molitor after exposure to static magnetic field (50 mT).

Material and methods: The experimental groups were: Control (kept 5 m from the magnets), groups which pupae and adults were placed closer to the North pole, or closer to the South pole of magnetic dipole. The pupae were exposed to the magnetic field until the moment of adult eclosion. The pupa-adult development dynamics were recorded daily. Subsequently, behaviour (distance travelled, average speed and immobility) of adults exposed to the magnetic field was monitored in a circular open field arena.

Results: Static magnetic field did not affect pupa-adult developmental dynamic of examined Tenebrio species. Exposure to magnetic field did not significantly change motor behaviour of T. obscurus adults. The changes in the motor behaviour of T. molitor induced by static magnetic field were opposite in two experimental groups developed closer to the North pole or closer to the South pole of magnetic dipole.

Conclusion: Static magnetic field (50 mT) did not affect on pupa-adult development dynamic of two examined Tenebrio species, but modulated their motor behaviour.     

Long-term continuous exposure to static magnetic field reduces popolysaccharide-induced cytotoxicity of fibroblasts

Purposes:?Lipopolysaccharide (LPS) is one of the major substances initiating the immune host response in microbial infections that results in cytotoxicity. In terms of treatment of the immune response, research has been conducted on physical environments that can reduce LPS-induced damage. In this experiment, a long-term continuous static magnetic field (SMF) was used as a physical resource to reduce LPS-induced immune host response.

Materials and methods:?Cultured fibroblasts were challenged with LPS to initiate an inflammatory reaction. Cell viability and various proinflammatory cytokine levels were detected and compared between SMF and sham-exposed groups.

Results:?Our in vitro study revealed that, with LPS challenge, fibroblasts continuously exposed to a 0.4-T SMF for 12 h demonstrated higher cell viability compared to unexposed analogs. From cytokine test, the levels of LPS-induced interleukin-1β (IL-1β) in the SMF-exposed groups were significantly lower relative to their unexposed counterparts (p < 0.05). By contrast, SMF exposure tended to increase the level of LPS-induced IL-1 receptor antagonist (IL-1Ra) and IL-6.

Conclusions:?Our results suggest that SMF stimulation inhibits LPS-induced cytotoxicity through reduction of proinflammatory cytokines and increase in anti-inflammatory cytokines of NIH-3T3 cells.     

Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons

Purpose: Although magnetic fields have significant effects on neurons, little is known about the mechanisms behind their effects. The present study aimed to measure the effects of magnetic fields on ion channels in cortical pyramidal neurons.

Materials and methods: Cortical pyramidal neurons of Kunming mice were isolated and then subjected to 15?Hz, 1?mT square wave (duty ratio 50%) magnetic fields stimulation. Sodium currents (I_Na), transient potassium currents (I_A) and delayed rectifier potassium currents (I_K) were recorded by whole-cell patch clamp method.

Results: We found that magnetic field exposure depressed channel current densities, and altered the activation kinetics of sodium and potassium channels. The inactivation properties of I_Na and I_A were also altered.

Conclusion: Magnetic field exposure alters ion channel function in neurons. It is likely that the structures of sodium and potassium channels were influenced by the applied field. Sialic acid, which is an important component of the channels, could be the molecule responsible for the reported results.     

Long-term exposure of cockroach Blaptica dubia (Insecta: Blaberidae) nymphs to magnetic fields of different characteristics: effects on antioxidant biomarkers and nymphal gut mass

Abstract

Purpose: The main goal of this study was to analyze the long-term effects of static (SMF) and extremely low-frequency magnetic field (ELF MF) on nymphal gut mass and antioxidant biomarkers in this tissue of cockroach Blaptica dubia.

Materials and methods: One-month-old nymphs were exposed to magnetic field (MF) for 5 months in three experimental groups: control, exposure to SMF (110?mT) and exposure to ELF MF (50?Hz, 10?mT).

Results: The gut masses of the MF groups were significantly lower when compared to control. Superoxide dismutase (SOD) and catalase (CAT) activities were markedly higher than for the control and the differences between the MF groups were statistically significant only for SOD. The applied MF had no effect on total glutathione (GSH) content. Glutathione reductase (GR) and glutathione S-transferase (GST) activities were significantly lower in both MF groups in comparison to the control. There was a significant difference between MF groups for GR activity. Principal Component Analysis (PCA) showed that CAT and GST were the main factors contributing to the differentiation of the control group from the treated experimental groups along PCA 1, and SOD and GR along PCA 2. PCA revealed clear separation between experimental groups depends on antioxidant biomarker response.

Conclusion: The applied magnetic fields could be considered a potential stressor influencing gut mass, as well as examined antioxidative biomarkers.     

Sulfonyl chromen-4-ones (CHW09) shows an additive effect to inhibit cell growth of X-ray irradiated oral cancer cells,involving apoptosis and ROS generation

Abstract

Purpose: This study evaluates the growth inhibiting potential of our previously described sulfonyl chromen-4-ones (CHW09) compound in X-ray irradiated oral cancer cells.

Materials and methods: The growth inhibiting effect and mechanism of combined CHW09/X-ray treatment was examined by analyzing cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), and DNA damage.

Results: Individual treatments of CHW09 (10?μg/mL) and X-ray irradiation (12?Gy) slightly decreased cell viability of oral cancer Ca9-22 (87.25% and 86.54%) and CAL 27 (80.00% and 74.01%) cells and normal oral HGF-1 cells (92.76% and 87.56%) at 24?h-MTS assay, respectively. In a combined treatment (CHW09/X-ray), the cell viability in Ca9-22 and CAL 27 cells was significantly decreased to 73.48% and 59.07%, whereas HGF-1 cells maintained 84.97% viability in 24?h-MTS assay. For CAL 27 cells, both 72?h-MTS assay and clonogenic assay showed that CHW09/X-ray resulted in more growth inhibition than other treatments. Intracellular ROS levels of CHW09/X-ray were higher than for CHW09, X-ray and control. CHW09/X-ray and X-ray alone had higher G2/M arrest than the control and CHW09 alone. Moreover, flow cytometry and western blotting showed that CHW09/X-ray treatment caused higher apoptosis levels. Levels of H2A histone family member X (γH2AX)-based DNA damage and 8-oxo-2′-deoxyguanosine (8-oxodG)-oxidative DNA damage of CHW09/X-ray were higher than for CHW09, X-ray and control.

Conclusion: CHW09/X-ray treatment had additive growth inhibiting effects against X-ray irradiated oral cancer cells, partly attributing to apoptosis and ROS generation.