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. 2026 Jan 24;16:6163. doi: 10.1038/s41598-026-37373-6

Efficient apoptosis via double-strand breaks of DNA in human epithelial cells induced by non-thermal effect of high-field broadband terahertz waves

Huan Zhou 1,2,3,4, Xingyi Wei 5, Xiao-Yu Peng 1,2,✉, Yangjiuzhou Gou 1,2, Zhang Yue 1,2, Julia Li Zhong 3, Jing Li 6, Cheng Zhang 6, Xi Zhang 6, De-Sheng Pei 5,✉
PMCID: PMC12905293  PMID: 41580463

Abstract

Induction of apoptosis is a primary strategy in cancer therapies in clinical oncology. Chemical or biological stimuli generally result in drug or agent resistance and damage to the immune system. Current physical stimuli have strong side effects, such as radiation injury or collateral thermal damage, due to the inflammation from the necrosis. Therefore, a new physical non-inflammatory therapy based on a non-thermal effect is highly desirable. Here, we report highly efficient apoptosis, reaching nearly 99% in breast cancer cells, one of the human epithelial cells, induced by the non-thermal effect of high-field (average field strength of ~ 5.87 MV/cm) broadband terahertz (THz) waves via double-strand breaks of DNA damage after 4 h of irradiation. This is accompanied by 11 highly differentially expressed genes related to apoptosis and significant morphological changes in the cells. These results suggest that the non-thermal effect of high-field broadband THz waves could be used to develop a potential novel, noninvasive, and non-inflammatory therapy for human superficial tumors.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-37373-6.

Keywords: High-field terahertz waves, Non-thermal effect, Apoptosis, DNA damage, Double-strand break, Human epithelial cells.

Subject terms: Biophysics, Cancer, Oncology

Introduction

Induction of apoptosis (programmed cell death) is one of the main therapeutic strategies in clinical oncology1–3. Chemical or biological stimuli generally result in drug or agent resistance and damage to the immune system4. Physical stimuli like X-ray5 or radioactive particles in brachytherapy6 have strong side effects (radiation injury) on the normal tissues around the tumor. Other physical stimuli applied in thermal therapies, such as radiofrequency ablation7, microwave ablation8, ultrasound ablation9, photothermal therapy10, laser ablation11, and cryoablation12, the current minimally invasive treatment methods, would induce necrosis and result in inflammation and collateral damage13–15. Therefore, a new physical, noninvasive, and non-inflammatory therapy based on non-thermal effects is highly desirable. Fortunately, a glimmer of light has been seen from recent research progress16,17 that strong pulsed THz waves may be used to induce the double-strand breaks of DNA, which would possibly induce apoptosis of tumor cells.

THz radiation typically refers to electromagnetic waves with frequencies ranging from 0.1 to 10 THz or wavelengths between 3 mm and 0.03 mm. Because the swing, twist, vibration, and rotation modes of biomacromolecules fall in the THz band18–21, when biological tissues or cells are irradiated with strong THz waves, these motion modes would be excited to trigger the linear or even nonlinear resonances and bring a series of biological effects22–28, including gene up- or down-expression16,22,24 and even DNA damage16,17. Possibly due to the insufficient strength of the THz wave or the insufficient THz irradiation dose (It can be defined as the average intensity of THz beam times the irradiation duration) to produce a sufficiently strong nonlinear resonance effect, however, the apoptosis of cancer cells due to DNA damage induced by strong pulsed THz waves has not been reported16,17,26, let al.one the irradiation threshold leading to apoptosis and the apoptotic mechanism. Furthermore, because both the stretching and bending vibration modes of the hydrogen bonds among water molecules fall in the THz band29,30, both continuous and pulsed THz waves would be absorbed strongly by the water in the cancer cells or tissues and result in some thermal effects23 that would bring the necrosis and inflammation31. Therefore, how to stimulate apoptosis without causing necrosis would be another challenge in realizing a non-inflammatory cancer therapy using THz waves. Here, we present the non-thermal biological effect of high-field (≥ 4 MV/cm) broadband pulsed THz waves on human epithelial cells, in which their DNA molecules were damaged and apoptosis was triggered efficiently mainly by the nonlinear resonance effect, showing a different apoptosis signaling pathway from the current known p53-dependent, exogenous, and endogenous apoptotic pathways.

Results

High-field THz waves induce efficient apoptosis by a non-thermal effect

To explore the dose threshold of THz radiation able to induce apoptosis, we investigate the apoptosis rates of one of the human epithelial cells, the human breast cancer (MCF-7) cells (cultured in 96-well plate) measured with flow cytometry after irradiated by high-field (average field strength of 5.46 MV/cm) THz waves with the average intensity of ~ 496.8 mW/cm2 and the converging THz beam with the diameter of ~ 10 mm (larger than the inside diameter of 6 mm of each well) at a repetition rate of 1 kHz in a thermostat at the physiological temperature of 37 °C for different irradiation time from 0 to 4 h. The experimental setup is shown in Fig. S1. Since the cell number of the monolayer cells growing at the bottom from a single well of a 96-well plate was insufficient for flow cytometric analysis, cells from four independent experimental replicates were put together for each measurement. We find that at least 1 h of irradiation time under the above-mentioned conditions is necessary for the induction of the evident apoptosis of MCF-7 cells, and the total apoptosis rate increases with the irradiation time. The total apoptosis rate reaches ~ 70% after 3 h of THz irradiation (Fig. 1a and b). In more detail, the total apoptosis rate increases monotonously with irradiation time. We noticed that the temperature of the cancer cells after 4 h of irradiation increased by ~ 3 °C compared with that of controls (Fig. 1c), however, the necrosis rate is always smaller than ~ 4% after THz irradiation for 1 to 4 h (Fig. 1a). This means that the thermal effect due to ~ 3 °C increment in temperature would not induce evident necrosis in MCF-7 cells.

Fig. 1.

Fig. 1

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Apoptosis of cancer cells induced by non-thermal effects of high-field THz waves vs. that induced by heat. a Apoptosis and necrosis rates of MCF-7 cells in 0.05 mL media cultured in a 96-well plate measured with flow cytometry after irradiated by high-field (average field strength of 5.46 MV/cm) THz waves with an intensity of ~ 496.8 mW/cm2 at a repetition rate of 1 kHz in a thermostat at the temperature of 37 °C for different irradiation time from 0 to 3 h. b Dependence of the total apoptosis rate and necrosis of MCF-7 cells on the irradiation time of high-field THz waves, corresponding to a. c The temperature of 4 h THz irradiated MCF-7 cells vs. that of the controls in a was measured by a thermal imager. d The apoptosis and necrosis rates of human breast cancer cells at different incubation temperatures from 37 to 44 °C in the thermostat. e Dependence of apoptosis and necrosis rates of MCF-7 cells on the temperature of the thermostat induced by heat from 37 to 44 °C for 4 h.

To evaluate how much the temperature of the irradiated cancer cells would induce necrosis and apoptosis, we performed separate experiments to investigate the dependence of apoptosis and necrosis rates of MCF-7 cells on the temperature in the thermostat induced by heat from 37 to 44 °C for 4 h. We find that the heat would not induce both apoptosis and necrosis (Fig. 1d and e) if the temperature is below 41 °C. When the temperature is higher than 41 °C, a small apoptosis rate of less than 10% is observed. However, once the temperature is increased over 43 °C and up to 44 °C, a sudden increase in the necrosis rate from ~ 2.53% to 68.3% is observed. These results indicate that if one wants to avoid the necrosis that would bring the inflammation, the temperature of the THz irradiated MCF-7 samples should be controlled to be less than 43 °C. The temperature of the THz irradiated cells shown in Fig. 1c is less than 40 °C, suggesting that all the apoptosis of the MCF-7 cells in Fig. 1a could be attributed mainly to the non-thermal effect of the high-field THz waves.

To obtain a higher apoptosis rate, we increased the intensity of the THz source up to ~ 573 mW/cm2 (corresponds to 4.86 MV/cm of the average THz field strength at the 10 mm diameter of the converging THz beam), which is currently the strongest intensity of the THz source in our lab and keep other experimental conditions as the same of in Fig. 1a. Due to the inside diameter of each well of the 96-well plate is 6 mm, the corresponding average field strength of the converging THz beam within the diameter of 6 mm is estimated to be ~ 5.87 MV/cm. We observe that the early and late apoptosis rates reach to 69.7% and 28.9%, respectively (Fig. 2a). The total apoptosis rate reaches to 98.6% (Fig. 2b), while the total apoptosis rate in controls is near 0. Please note that the necrosis rate is only 0.066% in the THz irradiated MCF-7 cells and the temperature of the MCF-7 cells after 4 h of THz irradiation increased ~ 6 °C from ~ 37 °C to ~ 43 °C (Fig. 2c). These results indicate that only the thermal effect due to increment in temperature up to 43 °C would still not induce evident necrosis in MCF-7 cells, corresponding to the highest temperature limit of 43 °C for the necrosis happening threshold shown in Fig. 1e, suggesting that all the apoptosis of the MCF-7 cells from Fig. 2a could be attributed mainly to the non-thermal effect of the high-field THz waves.

Fig. 2.

Fig. 2

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Efficient apoptosis of cancer cells induced by the non-thermal effect of high-field THz waves. a The apoptosis rate of MCF-7 cells in 0.05 mL media cultured in a 96-well plate irradiated by high-field (average field strength of ~ 5.87 MV/cm) THz waves with an intensity of ~ 573 mW/cm2 at a repetition rate of 1 kHz in a thermostat at a temperature of 37 °C. b Statistics of the apoptosis rates of the THz irradiated cells vs. that of the controls in the case of a. c The temperature of 4 h THz irradiated MCF-7 cells vs. that of the controls measured by a thermal imager in the case of a. d Dependence of the temperature of the MCF-7 cells irradiated by high field (average field strength of 5.46 MV/cm) THz waves with an intensity of ~ 496.8 mW/cm2 for 4 h on the repetition rate of THz pulses. e The apoptosis rate of MCF-7 cells for 4 h at the repetition rate of 700 Hz of the THz pulses (keep the same average field strength of 5.46 MV/cm) vs. those of controls. f Statistics of the apoptosis rates of the THz irradiated cells vs. those of the controls in the case of e.

In the above experiments, the THz irradiation duration is up to 4 h, which is a relatively long time for practical application. To reduce the irradiation time and obtain a high apoptosis rate at the same time, the intensity of THz waves must be increased. However, this increment in THz intensity would result in the temperature increase exceeding the threshold of 43 °C, which would bring the necrosis of the MCF-7 cells. We propose a strategy of reducing the repetition rate of the THz waves to ensure the temperature does not exceed the threshold of 43 °C if the THz field strength is increased. In our experiment, though the strongest average THz field strength is 5.46 MV/cm and could not be improved due to the limited highest energy of the fs pump laser system, we observed that the temperature of the sample was decreased monotonously from ~ 40 °C to ~ 38 °C with the decrease of the repetition rate of the THz waves from 1 kHz to 700 Hz but kept the same average strength of the THz field as 5.46 MV/cm (Fig. 2d). Even in the case of a repetition rate of 700 Hz, the apoptosis rate of MCF-7 cells can still reach to 56.7% (Fig. 2e and f). It could be extrapolated that the sample temperature could be controlled by reducing the repetition rate of the THz waves, but without the sacrifice of the high apoptosis rate if a stronger THz source is used.

High-field THz waves induce microscopic alterations in apoptotic cells

The results in Fig. 2a and b show that most of the MCF-7 cells after 4 h of THz irradiation tend to early apoptosis. To further validate this early apoptosis, we investigate the microscopic alterations in cell structure using transmission electron microscopy (TEM), which is the gold standard for assessing apoptosis. The MCF-7 monolayer cells were cultured in a 48-well plate in which each well has an inside diameter of 10 mm. We observed normal morphology and microstructure of the control cells (Fig. 3a and b) but distinct alteration of the morphology and microstructure of the cells that have undergone apoptosis after 4 h irradiation by high-field (average field strength of 4.52 MV/cm) THz pulses (corresponding to the average intensity of 496.8 mW/cm2 at 1 kHz repetition rate) (Fig. 3c and d). We find that the nuclei are irregular in shape, the chromatin is condensed, the nucleoli are clearly visible, and the boundary between the nucleus and cytoplasm is blurry. The rough endoplasmic reticulum (ER) is a parallel double-layer membrane with a narrow space in the cyst cavity and ribosomes attached to the cytoplasm. We note that the cell membrane is still continuous and intact. These characteristics of the microstructure suggest that these THz-irradiated cells are either mildly apoptotic or in an early apoptotic state, consistent with the results in Fig. 1a and b, which show that most cancer cells are in the early stages of apoptosis after 4 h of THz irradiation.

Fig. 3.

Fig. 3

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Microscopic changes of apoptotic cancer cells and DNA damages induced by non-thermal effects of high-field THz waves. The MCF-7 monolayer cells were cultured in a 96-well plate in which each well has an inside diameter of 6 mm. a Electron microscopic photograph of a typical MCF-7 cell of controls and its partially enlarged one (×25000) is shown in b. c Electron microscopic photograph of a typical early apoptotic MCF-7 cell after 4 h high-field (average field strength of 5.52 MV/cm) THz irradiation with the average intensity of 496.8 mW/cm2 at 1 kHz repetition rate of THz pulses and its partially enlarged one (×25000) in d. Nucleus (N), mitochondria (Mi), rough endoplasmic reticulum (RER); Chromatin agglutination (Inline graphic), autophagy (↑), cytoplasmic concentration (Inline graphic ), cell membrane continuity and integrity (↑) ×25,000. e DNA damage assays of MCF-7 cells in a 24-well plate irradiated by high-field (average field strength of 4.86 MV/cm) THz pulses with the average THz intensity of 573 mW/cm2 at a repetition rate of 1 kHz vs. the controls and that of the incubated MCF-7 cells in a water bath at 39.5 °C. The DNA-damaged (Double-strand breaks) cells were stained with γ-H2AX (green), and the staining of nuclei was performed with DAPI (blue). f The statistical DNA damage rates taken from e. g Statistical DNA damage rates of the THz irradiated MCF-7 cells at different zones of the converging THz beam: 0 ≤ R1 ≤ 0.17 cm, 0.17 ≤ R2 ≤ 0.34 cm, 0.34 ≤ R3 ≤ 0.50 cm, corresponding to the average field strength of 5.87, 4.86, and 2.53 MV/cm, respectively.

High-field THz waves induce DNA damage in apoptotic cells

In our experiments, high-field THz waves may induce a fierce nonlinear resonance effect in the DNA molecules, resulting in DNA damage and subsequently triggering the apoptosis signaling pathway. To validate this speculation, we used the γ-H2AX produced by H2AX phosphorylation to reflect clearly the degree of DNA damage with double-strand breaks in the MCF-7 cells (cultured in a 24-well plate) irradiated for 4 h by high-field (average of field strength of 4.86 MV/cm) THz pulses (573 mW/cm2 at a repetition rate of 1 kHz). The typical images at the center of the THz irradiated area of the MCF-7 cell sample vs. that of the controls as well as the sample incubated in a water bath at 39.5 °C are shown in Fig. 3e. One can see that the content of γ-H2AX is very low in the untreated cells (controls) and in the MCF-7 cells incubated in water bath, since there is almost no green fluorescence staining in the nucleus (Fig. 3e). However, the content of γ-H2AX significantly increased in the THz-irradiated cells and the green fluorescence in the nucleus was significantly enhanced (Fig. 3e). The corresponding DNA damage rate with double-strand breaks of THz-irradiated cells at the center of the converging THz beam reaches to 38.58% (Fig. 3f), much higher than that (~ 2.5%) of the controls and the cells incubated in water bath. These results indicate that the heat stress at 39.5 °C would not induce the DNA damage but the high-field THz waves would have induce the DNA double-strand breaks. It would be the DNA double-strand breaks that lead to subsequent apoptosis of the cancer cells.

As a Gaussian distribution of the THz beam, the local intensity decreases from the center to the edge along the radial of the converging THz beam. We find that the closer to the THz beam center, the stronger the green fluorescence intensity in the nucleus would be (Fig. S2). The DNA damage rate reaches to 38.58%, 18.92%, and 5.51% at the different zones with their radial ranges of 0 ≤ R1 ≤ 0.17 cm, 0.17 ≤ R2 ≤ 0.34 cm, 0.24 ≤ R3 ≤ 0.50 cm from the center, respectively, corresponding to the average THz field strength of 5.87, 4.86, and 2.53 MV/cm, respectively, as shown in Figs. 3g and S2. These results indicate that the DNA damage rate with double-strand breaks depends on the strength of the THz field or the intensity of the THz waves.

Highly expressed target genes are related to the apoptosis

To demonstrate the signaling pathway or the apoptosis mechanism, firstly we performed RNA sequencing on the MCF-7 cells (cultured in a 48-well plate) after irradiated for 4 h under the same conditions (average of THz field strength of 4.86 MV/cm) in Fig. 3e. We screened out 125 up-regulated and 2 down-regulated differentially expressed genes for further analysis from the differentially expressed genes volcano plot (Fig. 4a), much different from Titova’s results16 of 201 genes up-regulated and 241 genes down-regulated after 10 min irradiation by THz pulses with the energy of 1.0 µJ. Secondly, we performed MSigDB enrichment analysis of annotated differentially expressed genes by the Phyper based on the Hypergeometric test. The top 20 MSigDB signaling pathways are shown in a bubble diagram (Fig. 4b). Thirdly, we retrieved 148 and 161 apoptosis genes from Which Genes and MSigDB databases, respectively. By merging with 127 differentially expressed genes in transcriptome sequencing and removing duplicates, we identified a total of 5 target apoptotic genes, namely EGR3, JUN, GADD45B, BTK, and TNF. Combining MSigDB signaling paths analysis, we located the target genes in the following 11 genes: EGR1, FOSB, JUN, SOCS3, ID2, GADD45B, EGR3, BTK, TNF, EGR2, and CXCL11, as shown in the clustering heatmap (Fig. 4c) and the core PPI network (Fig. 4d). The TPM levels of these 11 target genes were compared with those of the controls as shown in Fig. 4e, from which much higher expression of the THz irradiated cells over the controls are observed. To further validate these 11 highly expressed target genes, a quantitative RT-qPCR technique was used. The results are shown in Fig. 4f. One can see that the 11 target genes in the THz-irradiated cancer cells indeed exhibit much higher expression than those in the controls, consistent with the transcriptome sequencing results (Fig. 4e).

Fig. 4.

Fig. 4

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High-expressed target genes related to apoptosis. a The differentially expressed genes volcano plot from the MCF-7 cells (cultured in 48-well plates) irradiated for 4 h by high-field (average field strength of 4.86 MV/cm) THz pulses with an intensity of 573 mW/cm2 at a repetition rate of 1 kHz. b Bubble diagram of the top 20 of MSigDB signaling pathways. c The clustering heatmap of 11 differentially expressed target genes. d The core PPI network is composed of 11 target genes. e Quantitative RT qPCR validation for the 11 highly expressed target genes from the THz irradiated MCF-7 cells over the controls based on TPM. f Quantitative RT qPCR validation for the 11 highly expressed target genes from the THz irradiated MCF-7 cells over the controls based on the relative mRNA level normalized to β-actin.

High-field THz waves promote the expression of target proteins

To further explore the mechanism of apoptosis of the THz irradiated MCF-7 cells (cultured in a 48-well plate with 10 mm inside diameter of each well), we used immunofluorescence staining kits to detect the expression level of two typical proteins, GADD45B and c-Jun, that play important roles in the apoptosis process. They are picked up according to the RNA sequencing and quantitative RT-qPCR results (Fig. 4e and f, and Table S1). Compared the fluorescence images for GADD45B and c-Jun proteins of high-field (average field strength of 4.86 MV/cm) THz irradiated MCF-7 cells with those of the controls (Fig. 5a and c), we observed that high-field THz waves promoted the expression of both target proteins of GADD45B and c-Jun (Fig. 5b and d).

Fig. 5.

Fig. 5

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Expression of target proteins. Immunofluorescence staining kits were used to detect expression levels of the GADD45B and c-Jun proteins. FITC-marked MCF-7 cells (cultured in 48-well plates) show green fluorescence, while the nuclei stained with DAPI show blue fluorescence. The intensity of green fluorescence reflected the level of protein expression. a Fluorescence images for GADD45B proteins of high-field (average field strength of 4.86 MV/cm) THz irradiated MCF-7 cells stained with FITC (green), DAPI, and a merge of them vs. that of the controls. b Statistics of the relative fluorescence of the THz irradiated MCF-7 cells vs. that of the controls in the case of a. c Fluorescence images for c-Jun proteins of THz irradiated MCF-7 cells stained with FITC (green), DAPI, and a merge of them vs. that of the controls. d Statistics of the relative fluorescence of THz irradiated MCF-7 cells vs. that of the controls in the case of c.

Discussion

Physical stimuli like α, β, γ, X-rays, and ultraviolet radiation can induce DNA damage and result in cell apoptosis. It is generally thought that there are three main apoptosis pathways: the p53-dependent pathway, the exogenous death receptor pathway, and the endogenous mitochondrial pathway32,33. In the p53-dependent pathway, p53 is generally considered to be one of the main regulatory components in the process of radiation-induced apoptosis in mammalian cells34. The study by Bolaris showed that low-dose irradiation with X-rays induced the production of p53 in the embryonic rat brain, and the expression of p53 protein was significantly increased in all apoptotic cells, indicating the important role of p53 in inducing apoptosis in the developing brain35. In our case, however, the sequencing results from our experiments do not show an obvious alteration in the expression of the p53 gene (Table S1), suggesting that the apoptosis pathway induced by high-field THz waves may not be p53-dependent. Recent studies have also found more evidence for the p53-independent apoptosis pathway36.

In the exogenous death receptor apoptosis pathway, caspase is activated by an extracellular signal, while in the endogenous mitochondrial apoptosis pathway, caspases are activated by the release of caspase-activating factor from mitochondria. Caspases involved in apoptosis fall into two broad categories: Initiator and effector enzymes, which play roles upstream and downstream of death signal transduction, respectively. These caspases generally include Caspase-2, 3, 6, 7, 8, 9, and 10. However, in our RNA sequencing and RT-qPCR analysis results (Fig. 6a and b), no obvious activation of Caspase-3, 6, 7, 8, 9 as well as FAS, FADD, UQCRFS1 (Cytc), Apaf-1 genes are observed, suggesting that the DNA fragmentation (double-strand break damage) observed in our experiments may be not caused by caspase but by the fierce nonlinear resonance induced by high-field THz waves.

Fig. 6.

Fig. 6

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Transcriptome data of gene expression. The MCF-7 cells (cultured in 48-well plates) are irradiated for 4 h by high-field (average field strength of 4.86 MV/cm) THz pulses at a repetition rate of 1 kHz. a TPM level of 13 associated genes from the THz-irradiated MCF-7 cells vs. the controls. b The relative mRNA level of 13 associated genes from the THz-irradiated MCF-7 cells vs. the controls.

In recent years, more and more evidence shows that GADD45 and Jun’s genes are involved in the apoptosis pathway. The growth arrest and DNA damage-induced 45 protein (GADD45) family plays an important regulatory role in apoptosis37,38. Studies have shown that once DNA is damaged, GADD45 is rapidly produced in cells to participate in the DNA damage repair process and induce cell cycle arrest and apoptosis39–41. In addition, GADD45 is involved in the regulation of various cellular signaling pathways in tumor cells42,43. In the context of Ras-driven breast carcinogenesis, Gadd45α elicits its function through activation of the stress-induced c-Jun NH2-terminal kinase and p38 kinases, which contribute to an increase in apoptosis and Ras-induced senescence44. Zerbini et al. reported that the up-regulation of melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) would increase the expression of growth arrest and DNA damage-inducible 45 α (GADD45α) and GADD45γ genes45, which is sufficient to induce cancer cell apoptosis. The mechanism involves the activation of c-Jun NH2-terminal kinase (JNK) and the induction of growth arrest through the inhibition of the Cdc2-cyclin B checkpoint kinase. In other examples of the apoptotic response in the HepG2 human hepatoma cells46 or colon cancer cells47, GADD45 was triggered and upregulated expression. In our experiment, we find that the expression levels of GADD45B and C-jun proteins are increased significantly in THz-irradiated cells, consistent with the results of transcriptome sequencing (Fig. 4e and f). Furthermore, cyclin B1 is also highly expressed, indicating that the mechanism of apoptosis induced by high-field THz waves in human breast cancer cells is highly related to GADD45B and c-Jun, as well as cyclin B1.

On the other hand, activator protein-1 (AP-1) is considered an intracellular transcriptional activator, mainly composed of the proto-oncogene-encoded proteins Jun and Fos. It binds to DNA target sequences in the form of a homodimer or heterodimer complex. AP-1 regulates the expression of target genes in response to the effect of various stimuli on cells and is also implicated in regulating a variety of cellular processes, including cell apoptosis48. Rebolo’s research shows that overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Bcl-3 expression prevents apoptosis of IL-4-deprived TS1ab cells49. In Santos’s research, the early activation of the c-Fos and c-Jun genes occurred after intestinal ischemia-reperfusion injury, and these genes can act together to trigger cell proliferation and apoptosis50. In our experiments, we find that Fos and C-Jun (Fig. 4e and f), as well as BCL2L11 (Table S1), are highly expressed in the THz irradiated breast cancer cells, implying that AP-1 participates in the mechanism of apoptosis and induces the expression of apoptosis-promoting gene BCL2L11.

Oxidative stress directly or indirectly initiates DNA double-stranded breaks (DSBs) by producing excessive reactive oxygen species (ROS). During the apoptosis signaling triggered by this DNA damage, the activated DNA damage response pathway ultimately triggers both the intrinsic (mitochondria-dependent) and extrinsic apoptotic pathways to achieve programmed cell death. In the mitochondria, p53 upregulates Bax → Bax inserts into the mitochondrial membrane → releases cytochrome C → combines with Apaf-1 to form the aposome → activates Caspase-9 → initiates the Caspase cascade; while in the death receptor (i.e., extrinsic) pathway, oxidative stress can also upregulate Fas/FasL expression → activates Caspase-8 → activates Caspase-3 directly or through Bid cleav. Both signaling pathways involve the significant upregulation of the expression and activation of the Caspase family, especially Caspase-3. However, our experimental results in Fig. 6a and b show that the series of Caspase enzymes, including Caspase-3, were not activated, and the expression was even downregulated, indicating that apoptosis induced by high-field THz waves in our experiments is unlikely to be due to the DNA double-stranded breaks from the excessive ROS produced by oxidative stress. In our experiments, the temperature of cell samples did not exceed 43 ˚C, and the flow cytometry results after irradiation also showed that the apoptotic rate of breast cancer cells at this temperature mostly came from the non-thermal effects of THz waves, rather than the DNA damage caused by the oxidative stress from the thermal stress. Although high-field THz waves could potentially cause damage to the cell membrane, the damage to the cell membrane itself does not directly lead to DNA double-strand breaks. Of course, damage to the cell membrane may indirectly affect the stability of DNA. For example, damage to the cell membrane may lead to changes in the intracellular environment, such as changes in pH value, ion concentration, etc., which may increase the level of oxidative stress within the cell, but the above analysis ruled out oxidative stress as the main cause of DNA damage in our experiments. Therefore, the possibility of oxidative stress, active oxygen, and membrane damage leading to DNA double-strand breaks in breast cancer cells in our experimental results can basically be ruled out.

Physical stimuli, including high-energy photons (such as X-rays, γ-rays) and high-energy particles (such as high-energy proton beams or other high-energy heavy ion beams), can directly produce ionization damage to DNA molecules or oxidative stress reactions that cause their-strand breaks. Non-physical stimuli such as chemical drugs or biological agents can directly damage DNA molecules or cause DNA double-strand breaks by oxidative stress, and affect DNA repair mechanisms. In our experiments, we only used THz wave for irradiating cells without using any chemical drugs or biological agents, and the electric field of THz waves is not yet strong enough to directly cause ionization damage to DNA molecules, so we speculate that it might be due to the non-linear resonance caused by high-field THz wave with DNA molecules that leads to their double-strand break damage. The physical basis is as follows: (1) The existing theory and results21,24,51–55 show that DNAs have twisting and H-bond motions at 0–2 THz, and all of their bases have many absorption peaks in the entire THz band, which are the physical basis for the overall and local resonance of the DNA molecules with THz waves. The THz source used in our experiments is a super broadband (0.1–24 THz) high-field source, which can theoretically excite the resonances of all the above absorption peak frequencies, which will cause the overall and local violent vibration of DNA double strands, i.e., the non-linear resonance, which will lead to DNA double-strand break damage. (2) The stronger the field strength of THz waves, the stronger the vibration of DNA molecules caused, and the greater the possibility of their damage. The additional experimental results in Fig.S3 (corresponding to Fig. 3g) show that the damage rate of the DNA molecule’s double-strand break is exponentially increasing with the field strength of THz waves, which also verifies the above speculation. (3) Theoretical studies have shown that THz waves of a specific frequency with a field strength of only a few hundred kV/cm can trigger the collective base flipping dynamics DNA and cause a large separation of base pairs24, and the field strength of THz waves used in our experiment is one order of magnitude than this field strength, so the damage to DNA molecules would be more serious. (4) The use of strong field THz waves of specific frequencies can achieve the demethylation of DNA molecules through the local nonlinear resonances with methyls on the double strands of the DNA27.

Based on our experimental results and above analysis, we speculate that under our experimental conditions, after the broadband (0.1–24 THz) THz waves penetrated the cell membrane and cytoplasm, it was very likely to generate overall and local nonlinear resonances with DNA molecules of tumor cells, which led to DNA double strand breaks and thus might triggered a possible new apoptotic signaling pathway independent of p53 and caspases: activating the overexpression of the GADD45B gene, then GADD45B reduces nuclear levels of cyclin B1 and activates the level of transcription factor AP-1 dimer (c-Jun-FosB), further up-regulates the expression of apoptosis-promoting protein BCL2L11, thereby triggers the cell apoptosis. However, the speculation of nonlinear resonances inducing the DNA double-strand breaks need to be directly verified by more in-depth experiments in the future to carry out pump-probe experiments relying on high-field THz sources, and the judgment made by detecting the nonlinear spectroscopic response of THz wave (such as harmonic generation or distortion of absorption peaks). However, this potential new apoptotic pathway, which appears independent of p53 and caspases, requires future validation through protein-level analysis (e.g., Western blotting, activity assays).

Although our current research is still at the cellular level, due to the highly efficient apoptosis in the cancer cells could be triggered mainly by the non-thermal effect via controlling the repetition rate or the intensity of the high-field THz waves, our research provides a novel idea and pave a way for the next research on the tumor ablation at the animal models and further clinical trials. For the weak-field THz waves, their penetration depth in water or biological tissues was reported to be sub-millimeter56,57. In our experiment, we used high-field THz waves with their electric field strength over 4 MV/cm, much stronger than that used in previous works56,57. To check the penetrating ability of the high-field THz waves in biological tissues, we performed an additional experiment using a piece of pork with a skin thickness of ~ 2 mm and ~ 6 mm of fat (~ 8 mm thickness in total) placed in the same position as shown in Fig. S1, but at the focus of the THz beam. We observed that after penetrating 8 mm of pork tissue, the intensity of the THz wave decreased from ~ 280 mW/cm² to 5.7 mW/cm², indicating that in biological tissues, high-field THz waves have a much longer penetration depth than weak-field THz waves. However, the attenuated intensity of 5.7 mW/cm² is insufficient to induce cellular apoptosis, indicating that the disadvantage of the limited penetration of the THz wave could be an advantage for the tumor ablation because the superficial tumor could be ablated layer by layer with the THz beams, but without damage to the normal tissues behind the tumor. If ablation of deep-seated tumors is required, future improvements in pump laser intensity to further increase the intensity of the THz wave will be necessary.

In conclusion, we observed efficient apoptosis (up to ~ 99%) in human epithelial cells via double-strand breaks of DNA damage induced by the non-thermal effect of high-field THz waves. We speculate that a possible p53- and caspase-independent apoptotic signaling pathway might be activated. The results suggest that the broadband high-field THz waves may provide a novel strategy for future non-invasive, non-thermal, and non-inflammatory physical therapy for superficial human tumors.

Methods

Cell culture

Human breast cancer MCF-7 cells (Procell Life Science & Technology Co. Ltd., CL-0149) were maintained in Advanced RPMI 1640 medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 10 mM Hepes, 100 units/mL penicillin, and 100 µg/mL streptomycin. Before the THz irradiation, the cancer cells were seeded into 96-well, 48-well, or 24-well cell culture plates at optimized densities suitable for each plate format, allowing them to form adherent monolayers on the well bottoms. Before THz irradiation on the subsequent day, the seeded cells were incubated overnight to ensure proper attachment and stabilization. Cell culture plates, including 96-well, 48-well, and 24-well polystyrene (PS) plates, were purchased from Corning Incorporated (USA). They were used in all experiments because of their minimal attenuation of THz waves (< 5%).

THz irradiation system

We used a self-built high-field (depending on the pulse energy and the spot size of the focused THz beam on the cells) broadband (0.1–24 THz with the central frequency of ~ 12 THz) pulsed THz source from laser-liquid interaction pumped by a Ti: sapphire laser system (12 mJ, 40 fs) at an adjustable repetition rate (from 1 Hz to 1 kHz) for the cell irradiation (Fig. S1a). This THz source could deliver a strong THz pulse with the highest average intensity Iavg= 573 mW/cm2 at a repetition rate of 1 kHz. We delivered the THz beam into a thermostat through an entrance window made of high resistance silicon wafer, focused it upward into the air above the well plate and irradiated the adherent grown cancer cells on the bottom of the well cell culture plate with a ~ 10 mm diameter of the converging THz beam by an off-axis parabolic mirror (OAPM) with a 90-degree turning angle (Figs. S1b and S1c) to collect enough THz-irradiated cells for the measurement of apoptosis rate by flow cytometry. The temperature in the thermostat was set constant ~ 37 °C, the physiological temperature of the cells. Non-irradiated cells cultured in other wells from the same well cell culture plate served as parallel controls, co-cultured with irradiated samples within the same time. To avoid the potential interference from the scattered THz waves or possible thermal conduction from the THz-irradiated samples, irradiated and control samples were separated by two empty wells. Cell viability and morphology were monitored by an electron microscope (JEM-1400FLASH, JEOL).

Calculation of the average field strength of the THz pulses

The average intensity Iavg of THz waves can be defined as the total energy of the THz pulses per unit time per unit area. If the repetition rate of the THz waves is k, meaning the THz source delivers k THz pulses in 1 s, we have Iavg = k*Inline graphic/S, where Inline graphic denotes the energy of a single THz pulse, S denotes the cross-sectional area of the THz beam. As a Gaussian THz beam, the average electric field strength Eavg at the 10 mm diameter of the converging THz beam can be calculated according to the equation: Eavg = [Iavg/(ε0 c)]1/2, in which ε0 denotes vacuum dielectric constant (ε0 = 3.85 × 10− 14 F/cm) and c is the speed of light in vacuum (c = 3 × 1010 cm/s), respectively. We obtain Eavg = ~ 4.86 MV/cm when Iavg= 573 mW/cm2 while the strongest peak electric field strength at the center of the converging THz beam is Emax ≈ (2)1/2Eavg = 6.87 MV/cm, and the electric field strength at the edge of the Gaussian THz beam is Eedg= Emax/e = 2.53 MV/cm. In the case of a 48-well plate, the inside diameter of each well is 10 mm, exactly the same diameter as the converging THz beam irradiating at the bottom of each well. In the case of a 96-well plate, the inside diameter of each well is 6 mm, less than the converging THz beam with a diameter of 10 mm. Thus, the average electric field strength in the THz irradiated area with diameter ≤ 6 mm falls in the range of 4.86 MV/cm ≤ Eavg≤6.87 MV/cm, which is around 5.87 MV/cm. In the case of a 24-well plate, the inside diameter of the well is 15.5 mm, larger than the converging THz beam with a diameter of 10 mm, only the center area with a diameter ≤ 10 mm can be irradiated by the THz beam. We use different well cell culture plates for different measurements.

Measurement of the cell temperature

Considering that the thin culture media would affect the accurate reading of the cell temperature and a Gaussian THz beam would bring a Gaussian distribution of temperature in the THz-irradiated samples, as well as to avoid damaging the cells, the liquid thermometer and thermocouple would not be suitable for use to measure the cell temperature. Here, a non-invasive technique with a visual IR camera (SMART SENSOR, ST8450, 0.05 °C of thermal sensitivity) was used to measure the temperature of the cells before, during, and after THz irradiation. The visual IR camera was placed 1 cm above the well plate. This infrared thermography has become the gold standard for non-contact thermal monitoring in such experimental paradigms, precisely because it circumvents cellular perturbation while maintaining temperature accuracy. With this technique, we can obtain the entire temperature distribution of the sample irradiated by a Gaussian THz beam. Though the IR camera had been calibrated before leaving the factory, for the sake of caution, we performed a separate experiment to validate our IR camera by comparing the temperature measurement results from the same sample after heating measured by our IR camera with those measured by a liquid thermometer and a thermocouple. The result showed the measurement by our IR camera was consistent with that measured by the liquid thermometer and thermocouple.

Flow cytometric assays for cell apoptosis

Since conventional CCK assays can only permit discrimination of live and dead cells, we employed flow cytometric techniques to classify the cells into early apoptotic cells, late apoptotic cells, and necrotic cells. Apoptosis was evaluated after the cells were irradiated by high-field THz waves using the APC Annexin V apoptosis detection kit with Propidium Iodide (PI), as per the manufacturer’s instructions (Biolegend, 640932). Since the cell number (typically 1–2 × 10⁴ cells) of the monolayer cells growing at the bottom per well of a 96-well plate was below the detection threshold of flow cytometry (at least 1–5 × 10⁵cells/sample), cells from four independent experimental replicates were put together for apoptosis measurement. This assay relies on two key interactions: APC Annexin V binds to phosphatidylserine residues exposed on the membrane surface of cells initiating apoptosis, while Propidium Iodide (PI) intercalates with cellular DNA exclusively in cells with fully disrupted membranes. The staining protocol was performed as follows: Harvested cells were collected, washed, and resuspended in 100 µL of Annexin V Binding Buffer. Subsequently, 5 µL of APC Annexin V and 10 µL of Propidium Iodide Solution were added to each sample, which were then incubated for 15 min at 25 °C in the dark. After staining, 400 µL of Annexin V Binding Buffer was added to each tube to dilute the samples. The proportion of apoptotic cells was quantified using a BD Accuri C6 flow cytometer (BD Biosciences, Ann Arbor, MI) with instrument parameters optimized for this specific assay.

DNA damage assays

Human breast cancer cells were uniformly cultured on poly-L-lysine-coated cell climbing tablets in a 24-well plate. DNA Damage Assays were evaluated after irradiation of the cells by the high-field THz waves using the DNA damage assay kit by γ-H2AX immunofluorescence, as per manufacturer’s instructions (Beyotime, C2035S). To confirm the occurrence of DNA damage, immunofluorescence staining was employed to detect γ-H2AX—a well-recognized marker for DNA damage, which corresponds to phosphorylated H2AX. When DNA double-strand breaks occur, proteins belonging to the Phosphatidylinositol 3-kinase-related kinases (PIKKs) family, specifically ATM (Ataxia-telangiectasia mutated protein) and ATR (ATM and RAD3-related protein), mediate the phosphorylation of serine 139 on the H2AX molecule. This phosphorylation event gives rise to phosphorylated H2AX, and the expression level of γ-H2AX (the product of H2AX phosphorylation) serves as a reliable indicator to quantitatively assess the extent of DNA damage. The experimental procedure was carried out as follows: First, the cells were rinsed once again with phosphate-buffered saline (PBS) to remove residual culture medium. Next, a fixative solution was added to the cells, which were then incubated for 15 min to preserve their structural integrity. After fixation, the cells were sequentially treated with blocking buffer, γ-H2AX rabbit monoclonal antibody (γ-H2AX rabbit mAb), and anti-rabbit 488 secondary antibody; each incubation step was conducted at room temperature for 1 h to ensure sufficient reaction. Following the antibody incubations, the nuclear stain 4’, 6-diamidino-2-phenylindole (DAPI) was added to the cells, which were further incubated for 5 min at room temperature to label the cell nuclei. Subsequently, an appropriate volume of antifade mounting medium was added dropwise to the samples to prevent fluorescence quenching, and the samples were sealed with a coverslip. Finally, the stained cells were observed and analyzed using a fluorescence microscope. After staining, γ-H2AX emitted green fluorescence with an excitation wavelength (Ex) of 495 nm and an emission wavelength (Em) of 519 nm, while cell nuclei stained with DAPI emitted blue fluorescence with an Ex of 364 nm and an Em of 454 nm.

RNA sequencing (RNA-seq)

Total RNA was isolated from the cells using Trizol reagent (Ambion, 343902) in strict accordance with the manufacturer’s protocol. Subsequently, the purity, integrity, and concentration of the extracted total RNA were subjected to qualitative and quantitative assessments using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, MA, USA), respectively, to ensure the RNA samples met the quality requirements for subsequent experimental procedures. The construction of the RNA library and the subsequent RNA sequencing workflow were carried out as follows: First, random hexamers were used as primers to initiate reverse transcription, generating the first-strand cDNA. Following this, the second-strand cDNA was synthesized using the first-strand cDNA as a template. After the completion of double-strand cDNA synthesis, an A-Tailing Mix was added to perform end repair and A-tailing, and RNA Index Adapters were ligated to the cDNA fragments. cDNA fragments were amplified via PCR, and the products were purified with Ampure XP Beads and eluted into EB buffer. The quality of the purified library was verified using an Agilent Technologies 2100 Bioanalyzer to confirm that the fragment size, concentration, and integrity of the library were suitable for sequencing. The double-stranded PCR products that passed quality verification were heated to denature, and then circularized with the assistance of a splint oligo sequence to form single-strand circle DNA (ssCir DNA), which served as the final sequencing library. The final library was amplified using phi29 DNA polymerase to generate DNA nanoballs (DNBs), where each DNB contained more than 300 copies of a single molecular template. These DNBs were loaded onto a patterned nanoarray, and single-end 50-base reads were generated on the BGIseq500 sequencing platform to obtain raw sequencing data. For the processing of sequencing data: SOAPnuke software (v1.5.2) was employed to filter the raw sequencing data, removing low-quality reads, adapter sequences, and contaminated sequences to obtain high-quality clean reads. The clean reads were aligned to the reference genome using HISAT2 software (v2.0.4) and further aligned to the reference coding gene set using Bowtie2 software (v2.2.5). The expression levels of genes (represented as TPM values) were calculated using RSEM software (v1.2.12). A clustering heatmap was generated using pheatmap software (v1.0.8) based on the gene expression levels across different samples, providing a visual representation of the gene expression difference patterns. Differential expression analysis was performed using DESeq2 software (v1.4.5), with a false discovery rate (FDR)-adjusted P-value (q-value) ≤ 0.05 set as the threshold for screening differentially expressed genes. To explore the association between differentially expressed genes and changes in cell phenotype, MSigDB (https://www.gsea-msigdb.org/gsea/msigdb) enrichment analysis of annotated differentially expressed genes was conducted using the Phyper tool (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on the Hypergeometric test. The significance levels of the enriched pathways were corrected using the Bonferroni method, and a q-value ≤ 0.05 was used as the criterion for determining significantly enriched pathways.

Quantitative RT-PCR analysis

Total RNA isolation was performed using Trizol reagent following the manufacturer’s recommended protocol. The extracted RNA was subsequently reverse-transcribed into complementary DNA (cDNA) utilizing the Bestar® qPCR RT RT Kit (DBI Bioscience, DBI-2220). Real-time PCR amplifications were conducted with Bestar® SYBR Green qPCR master mix (DBI Bioscience, DBI2043) on a CFX Connect Real-Time-Time PCR Detection System (Bio-Rad, 1855201). All reactions were performed in triplicate to ensure reproducibility. Relative mRNA expression levels were determined using the comparative cycle threshold (Ct) method. Expression values were first normalized to the housekeeping gene β-actin, and then relative quantification was performed against the control group. The specific primer sequences used in this experiment are detailed in Supplementary Table S2.

Immunofluorescent staining

Due to the limitation in the number of THz-irradiated cells, we could not extract enough proteins for Western blot detection. Therefore, we utilized immunofluorescent staining for the detection of target proteins. Immunofluorescence staining kit (Anti-Rabbit IgG-FITC, Elabscience, E-IR-R323) and immunofluorescence staining kit (Anti-Mouse IgG-FITC, Elabscience, E-IR-R324) were used to detect GADD45B and C-Jun. Following fixation in 4% paraformaldehyde solution for 30 min to preserve cellular structure, the cells were permeabilized with 0.5% Triton X-100 for 15 min to facilitate antibody penetration. For nonspecific binding blocking, the cells were then incubated in normal goat blocking buffer for 30 min as a preprocessing step. Subsequently, the cells were transferred to a moisture-resistant, light-protected container and incubated with primary antibodies at 37 °C for 1 h. The primary antibodies used were GADD45B antibody (diluted at a ratio of 1:200) and C-Jun antibody (diluted at a ratio of 1:100). After the primary antibody incubation, the cells were further incubated in the dark for 1 h with appropriate fluorophore-conjugated secondary antibodies to enable signal detection. Specifically, goat anti-rabbit IgG (H + L) conjugated with fluorescein isothiocyanate (FITC) was used for GADD45B antibody staining, while goat anti-mouse IgG (H + L) conjugated with FITC was applied for c-Jun antibody staining. Finally, fluorescent signals were visualized and imaged using a fluorescence microscope.

Statistical analysis

Results of each experiment were obtained three times (n = 3 replicates), with three biological replicates used in our experiment, unless noted otherwise. The experimental data are expressed as the mean ± standard deviation (SD). Statistically significant differences between group means were determined by a two-way ANOVA test with Dunnett’s post hoc multiple comparison method and by the paired t-test, using OriginPro 9.0 and GraphPad Prism 8.0. A result of p < 0.05 was considered significant (*); p < 0.01 was considered extremely significant (**); p < 0.001 was considered extremely significant (***); p < 0.0001 was considered extremely significant (****).

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (9MB, docx)

Acknowledgements

We thank BGI-Shenzhen of China who supported the RNA sequencing. X.-Y. Peng would like to thank Prof. Mingxia He for her helpful discussion.

Author contributions

H. Zhou: Investigation, formal analysis, methodology, writing–original draft. X. -Y. Peng: Conceptualization, design of the THz source and its irradiation system, investigation, methodology, funding acquisition, supervision, resources, writing–original draft, writing–review and editing. X. Wei, Y. Gou, Z. Yue, J. L. Zhong, J. Li, C. Zhang, and X. Zhang: Methodology, resources. D. -S. Pei: Investigation, supervision, resources, funding acquisition, writing–review, and editing. All authors reviewed the final manuscript.

Funding

This work was supported by the NSAF (Grant No. U2030119), High-level Talents Project of Chongqing Medical University (No. R4014), and Chongqing Technology Innovation and Application Development: Sichuan-Chongqing Science and Technology Innovation Cooperation Program Project (CSTB2024TIAD-CYKJCXX0017).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Raw data have been deposited to the National Center for Biotechnology Information (NCBI) under the BioProject Accession Number PRJNA1354789.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Xiao-Yu Peng, Email: xypeng@cigit.ac.cn.

De-Sheng Pei, Email: peids@cqmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (9MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Raw data have been deposited to the National Center for Biotechnology Information (NCBI) under the BioProject Accession Number PRJNA1354789.

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