Short-Term Exposure to a 50 Hz Extremely Low-Frequency Electromagnetic Field (ELF-EMF) Leads to ROS-Mediated DNA Damage in Gynecological and Urological Cancer Cells In Vitro

TL;DR

Short-term exposure to a 50 Hz electromagnetic field increases DNA damage in gynecological and urological cancer cells through ROS production.

Contribution

This study shows that short-term ELF-EMF exposure induces ROS-mediated DNA damage in specific cancer cell lines.

Findings

Exposure to 50 Hz ELF-EMFs increased ROS levels in HeLa, ES-2, and DU-145 cancer cells.

ES-2 and DU-145 cells showed significant changes in DNA repair-related gene and protein expression after 30 min of exposure.

Short-term ELF-EMF exposure may offer a potential treatment strategy for gynecological and urological cancers.

Abstract

The effect of sinusoidal Extremely Low-Frequency Electromagnetic Fields (ELF-EMFs) on gynecological (HeLa, ES-2) and urological (DU-145) cancer cells was investigated. ELF-EMFs with a frequency of 50 Hz and a magnetic flux density of 1.3 mT were applied for 15 and 30 min. The experiment was conceptualized to investigate the in vitro short-term effects of ELF-EMFs on cell reactive oxygen species (ROS) formation, the levels of genes and proteins involved in DNA damage response, and epigenetic modifications. Here, we found that ELF-EMFs treatment leads to an elevation in the ROS levels that contribute to distinct scenarios in the studied cancer cells. The most prominent changes in the studied factors were found in ES-2 and DU-145 cells exposed to 30 min of ELF-EMFs. ES-2 cells exhibited upregulation of XRCC5 gene expression and elevated levels of several proteins: TNF-α, RAD51, APE1, XRCC1, and NSUN2. Diminished levels of BCL-2, HSP90, RAD51, and TNF-α, as well as overexpression of VIM and METTL3, were observed in DU-145 cells. In summary, we postulate that short-term exposure to 50 Hz ELF-EMFs may be a promising treatment strategy for gynecological and urological cancer cells.

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Full text

1. Introduction

Nowadays, humans are constantly exposed to extremely low-frequency electromagnetic fields (ELF-EMFs). ELF-EMF is non-ionizing radiation ranging from 1 to 300 Hz, generated by power lines and electrical devices. Most studies employ ELF-EMFs with a frequency of 50 and 60 Hz, as these parameters correlate with the environmental exposure [1]. The cellular response to ELF-EMFs depends on the intensity and strength of the magnetic field, the duration of exposure, the genetic and biological properties of cells [2], as well as the cell cycle and metabolic stage of the tested cell [2,3].

Recently, ELF-EMFs have attracted the attention of researchers due to their unique advantages, including non-invasive nature, tunable parameters, and the capacity to precisely modulate cellular and molecular processes [4,5,6,7]. The beneficial effects of ELF-EMFs have been exerted both in vitro and in vivo [8,9]. ELF-EMFs have been used to relieve pain [10,11,12], stimulate tissues such as muscle, nerve, heart, and retina [13,14], and accelerate wound healing [15]. Furthermore, ELF-EMFs have been suggested to be a potential strategy to fight trauma [16] and osteoporosis [17]. In recent years, ELF-EMFs have also been investigated as a potential tool in cancer therapy [18], as they can exert effects on key cellular processes, including angiogenesis, differentiation, apoptosis, immunity, and inflammation [4,19,20]. Moreover, ELF-EMFs can influence cell proliferation and elevate oxidative stress in cancer cells, inhibiting their growth and enhancing their sensitivity to chemotherapeutics, such as doxorubicin (DOX) [18], docetaxel [21], cisplatin, methotrexate, and ifosfamide [22], potentially enabling lower drug doses and reducing side effects. EMFs can improve the effectiveness of chemotherapy through multiple mechanisms, including increased drug uptake mediated by changes in membrane permeability [23], induction of mitochondrial dysfunction [24], and modulation of apoptosis-related gene expression [18]. Unlike conventional treatments such as chemotherapy or radiotherapy, ELF-EMFs have been suggested to selectively affect cancer cells, while leaving the normal cells intact [25]. Although most studies have shown that extremely low-frequency magnetic fields exhibit an inhibitory effect on cancer cells [26], some studies point to a stimulatory effect. For instance, Wolf and colleagues have shown that exposure to 50 Hz (0.5–1 mT) ELF-EMFs induced a dose-dependent increase in the proliferation of HL-60 leukemia cells [27]. Additionally, different groups reported that 50 Hz with the magnetic flux of 1 mT promotes the survival of cancer cells through the activation of the antioxidative and detoxification defense systems [28].

Despite promising results in cancer cells, the underlying mechanisms of ELF-EMFs remain incompletely understood, thus limiting their broader application as a therapy. Most evidence comes from preclinical studies (cell or animal model), and well-controlled human trials remain scarce [29]. Additionally, biological responses differ between cell types and tissues, complicating targeted therapy. Finally, the lack of standardized devices and treatment guidelines limits clinical adoption. Therefore, understanding the molecular mechanisms underlying the effects of ELF-EMFs on a broad range of cancer types is crucial in answering the question of whether ELF-EMFs have a positive or negative impact on cancer cells and whether ELF-EMFs can be applied as a possible treatment strategy. To answer these questions, we tested the effect of sinusoidal ELF-EMFs with a frequency of 50 Hz and intensity of 1.3 mT using gynecological cancer cell lines: HeLa (cervical cancer) and ES-2 (ovarian cancer), and urological cancer cell line: DU-145 (prostate cancer). As most of the studies focus on the long-term ELF-EMF exposure effect, we decided to determine whether a short-term exposure has any impact on the tested cells. Thus, the cells were subjected to an ELF-EMF exposure for 15 and 30 min, and next, the post-treatment effects were evaluated. The study examined changes in the levels of reactive oxygen species (ROS), as well as the gene and protein levels of factors involved in the DNA damage response (DDR) and epigenetic modifications.

2. Results

2.1. Extremely Low-Frequency Electromagnetic Fields (ELF-EMFs) Induce Reactive Oxygen Species (ROS)

The Fenton reaction is a process catalyzed by metal ions that converts H_2_O_2_ into toxic hydroxyl radicals [30]. Some studies have suggested that EMF works through the Fenton reaction, promoting free radicals in cells [31,32]. The presence of excessively high levels of reactive oxygen species (ROS) within the cell has been implicated in triggering cell death pathways, such as apoptosis and autophagy, leading to tumor suppression [33]. To explore the effect of ELF-EMFs exposure on ROS levels in gynecological and urological cells, we used Dihydroethidium (DHE)—a superoxide-responsive fluorescent probe [34]. The ROS levels decreased immediately after DHE probe removal (0 h time-point) in ES-2 cells treated with ELF-EMF for 30 min compared to CTR (** p < 0.01, Figure 1A). However, extension of incubation time led to a significant increase in the ROS levels following the DHE removal in tested cells exposed to ELF-EMFs for 15 min and 30 min, except the HeLa cells treated for 15 min (** p < 0.01, *** p < 0.001, Figure 1A). The ROS levels upregulation has also been observed in ES-2 cells exposed for 15 min and DU-145 cells exposed for 15 min and 30 min to ELF-EMFs, when compared to the 0 h time point (^#^ p < 0.05, ^##^ p < 0.01, Figure 1A).

Since ROS work as intracellular molecules that influence various cellular processes, we tested the mRNA levels of NFE2L2 (a gene encoding Nuclear factor erythroid-related factor 2 (NRF2)), a redox-sensitive transcription factor [35], and the protein levels of anti-apoptotic BCL-2, anti-oxidative HSP90 and HO-2, RNA-binding protein—HuR, inflammatory TNF-α, and cytoskeleton protein—Vimentin (VIM), in response to ELF-EMFs treatment. Our analysis revealed no statistically significant changes in the mRNA levels of NFE2L2 in HeLa and ES-2 cells upon the ELF-EMFs treatment (Figure 1B). A more prominent effect has been observed while testing protein levels of downstream factors. The expression of BCL-2 showed a statistically significant decrease only in DU-145 cells exposed to 30 min of 50 Hz ELF-EMF compared to CTR (** p < 0.01, Figure 1C). The levels of HO-2 significantly increased only in ES-2 cells treated with ELF-EMF for 30 min compared to 15 min of treatment (^#^ p < 0.05, Figure 1C). The levels of nucleic and cellular fractions of HSP90 were significantly downregulated in HeLa and DU-145 cells exposed for 15 and 30 min to ELF-EMF, except the HSP90 cellular fraction in DU-145 (** p < 0.01, *** p < 0.001, Figure 1C). In contrast, the HSP90 cellular fraction was upregulated in ES-2 cells treated for 15 min (** p < 0.01, Figure 1C); however, the observed effect was transient. No significant changes were determined in the levels of HuR in HeLa, ES-2, and DU-145 cells treated with 15 and 30 min of ELF-EMF compared to CTR (Figure 1C). Interestingly, the levels of TNF-α were significantly upregulated in ES-2 cells, while significant downregulation of TNF-α was observed in DU-145 cells after ELF-EMFs treatment compared to CTR (* p < 0.05, ** p < 0.01, *** p < 0.001, Figure 1C). We have also indicated a statistically significant upregulation of VIM in ES-2 cells exposed to ELF-EMFs for 15 min (** p < 0.01, Figure 1C). Elevated levels of VIM were also observed in DU-145 cells subjected to 30 min of ELF-EMFs (* p < 0.05, Figure 1C).

2.2. ELF-EMF Influences the Levels of DNA Repair Factors

Furthermore, we tested the levels of factors involved in the DNA damage response (DDR) in cells treated with ELF-EMFs. We determined the mRNA levels of H2AFX, OGG1, XRCC5, XRCC6, POLβ, and MAP1LC3B genes in control and ELF-EMF-treated cells. However, our analysis indicated a statistically significant increase only in XRCC5 mRNA levels upon a treatment of ES-2 cells for 15 and 30 min compared to CTR and 15 min of treatment with ELF-EMFs (*** p < 0.001, ^#^ p < 0.05, Figure 2A). No statistically significant changes in the mRNA levels of H2AFX, OGG1, XRCC6, POLβ, and MAP1LC3B have been determined in the tested cells in response to the treatment with ELF-EMFs (Figure 2A).

Next, we investigated the protein levels of RAD51, APE1, and XRCC1 in nucleic and cellular fractions of control and ELF-EMF-treated cells. We have shown a statistically significant increase in the levels of nucleic and cellular fractions of RAD51, APE1, and XRCC1 in ES-2 cells exposed to ELF-EMFs for 15 and 30 min, except the cellular fraction of APE1 and XRCC1 in cells exposed for 15 min (* p < 0.05, ** p < 0.01, *** p < 0.001, Figure 2B). An increase in the cellular XRCC1 levels has also been observed in HeLa cells exposed for 15 min to ELF-EMFs (* p < 0.05, Figure 2B). On the contrary, the levels of RAD51 nucleic and cellular fractions were significantly downregulated in DU-145 cells exposed to 15 and 30 min of ELF-EMFs compared to CTR (* p < 0.05, ** p < 0.01, Figure 2B).

2.3. Changes in the Expression of the m5C and m6A Methyltransferases in Response to ELF-EMFs

Our previous analysis indicated changes in the expression levels of factors responsible for epigenetic modifications in ARPE-19 and RGC-5 cells exposed to ELF-EMFs [13]. Therefore, we evaluated the levels of NSUN2 and METTL3 in cancer cells (Figure 3A,B). We have shown that the nuclear and cellular levels of NSUN2 were significantly upregulated in ES-2 cells after the treatment with ELF-EMFs for 15 and 30 min, compared to CTR (** p < 0.01, *** p < 0.001, Figure 3A). Similarly, the NSUN2 cellular fraction increased in HeLa cells exposed to ELF-EMF for 15 min compared to CTR (* p < 0.05, Figure 3A). An increase of cellular METTL3 levels has been observed only in DU-145 cells exposed for 30 min (** p < 0.01, Figure 3A).

3. Discussion

An enormous increase in the ELF-EMF sources to which people are daily exposed has recently been observed [36]. Therefore, interest in evaluating the effect of ELF-EMFs on human health and assessing their potential as a treatment strategy is expanding. Several studies indicated beneficial applications of ELF-EMFs alone or in combination with chemotherapy in cancer treatment [37,38,39]. However, these studies mostly focused on the correlation between long-term EMFs exposure and the effect on cancer cells [40,41], leaving the impact of short-term exposure to ELF-EMFs unrevealed. Our previous studies have shown that as little as 15 or 30 min of ELF-EMFs exposure causes changes in the intracellular signaling of non-cancerous cells [13]. Thus, we wondered whether the exposure to ELF-EMFs with the same parameters would have a curative effect on gynecological and urological cancer cells. In this study, we utilized cervical (HeLa), ovarian (ES-2), and prostate (DU-145) cancer cells to investigate the effects of ELF-EMFs with the frequency of 50 Hz, and magnetic flux density of 1.3 mT applied for 15 and 30 min.

While apoptosis is induced upon excessive production of ROS, moderate levels of ROS may contribute to cell proliferation, migration, and invasion. To maintain redox homeostasis, cells evolved a mechanism defense system, where the alternation in redox status can lead to transcriptional activation of pathways and enzymes contributing to ROS elimination [33]. Therefore, we first questioned whether the ELF-EMFs exposure of gynecological and urological cancer cells leads to an elevation of intracellular ROS levels. Our analysis has shown an enhanced production of ROS in ES-2 and DU-145 cells treated with ELF-EMFs for 15 and 30 min, compared to untreated CTR and the 0 h time point (** p < 0.01, *** p < 0.001, ^#^ p < 0.05, ^##^ p < 0.01, Figure 1A). An increase in ROS levels was observed 24 h post-treatment in HeLa cells exposed for 30 min to ELF-EMFs compared to CTR (** p < 0.01, Figure 1A). Similarly, data presented by our group have shown elevated levels of ROS in HeLa and DU-145 cells in response to continuous or pulsed 50 Hz electromagnetic fields [42]. Additionally, Yuan et al. have revealed that ROS levels increased shortly after exposure to 50 Hz, 5.1 mT EMFs and persisted until the next day in G401 and A549 cells [43]. Therefore, our data are in agreement with previous studies showing the role of ELF-EMFs in ROS induction [44]. Overall, we can suggest that short-term exposure to ELF-EMFs stimulates chronic ROS levels that may contribute to cancer cell death.

Subsequently, we verified whether ELF-EMF-induced ROS levels would persist or be attenuated by the antioxidant defense system. As NRF2 is a master regulator of the oxidant response, neutralizing the ROS balance [45], we further tested the mRNA expression of NFE2L2. Although we did not detect statistically significant changes in the mRNA levels of NFE2L2 in HeLa and ES-2 cells upon the ELF-EMFs treatment (Figure 2B), the differences in the expression levels of anti-apoptotic BCL-2 and some antioxidant enzymes were determined (Figure 1C). Our analysis indicated a decrease in the levels of BCL-2 in DU-145 cells treated with ELF-EMF for 30 min (** p < 0.01, Figure 1C). Similarly, diminished levels of BCL-2 have been observed in ZR-75-1 and MCF-7 breast cancer cells in response to EMFs treatment, however, with the higher parameters (200 Hz, 1 mT) for 24 h [4]. Our previous studies also indicated statistically significant downregulation of BCL-2 levels in RGC-5 cells right after exposure to ELF-EMFs for 15 and 30 min [13]. Conversely, exposure to sinusoidal ELF-EMFs did not influence the BCL-2 level in HeLa and ES-2 cells (Figure 1C). Similarly, no significant changes in BCL-2 protein levels have been found in K562 cells upon the ELF-EMF treatment [46]. Therefore, we can conclude that short-term exposure to ELF-EMF is sufficient to induce apoptosis only in prostate cancer cells.

Next, we examined the levels of HO-2 and HSP90 in ELF-EMF-mediated ROS conditions in gynecological and urological cells (Figure 1C). The levels of HO-2 significantly increased only in ES-2 cells treated with ELF-EMFs for 30 min compared to 15 min of treatment (^#^ p < 0.05, Figure 1C). No significant changes in the HO-2 levels have been observed in any of the tested cell lines compared to CTR (Figure 1C).

In cancer cells, Heat Shock Proteins (HSPs), especially HSP90, lead to cell survival, proliferation, and resistance to therapy [47]. HSP90 can also act as an antioxidant protecting cells against ROS-induced damage [48]. Conversely, upon the elevated ROS levels, HSP90 can be destabilized or cleaved, affecting its chaperone function [49]. Interestingly, our analysis revealed a significant downregulation of a nucleic fraction of HSP90 in HeLa and DU-145 cells treated with ELF-EMF for 15 and 30 min compared to CTR (** p < 0.01, *** p < 0.001, Figure 1C). Additionally, a cellular fraction of HSP90 was diminished in HeLa cells (*** p < 0.001, Figure 1C). On the contrary, the cellular fraction of HSP90 has been upregulated in ES-2 cells treated for 15 min with ELF-EMF (** p < 0.01, Figure 1C). This suggests that the ELF-EMFs induced ROS levels contribute to the HSP90 diminution in DU-145 and HeLa cells.

Hu antigen R (HuR) is an RNA-binding and stress-response protein that plays a crucial regulatory role under oxidative stress. Studies performed by Mehta et al. have revealed that silencing of HuR induces ROS production in triple-negative breast cancer cells [50]. Thus, we checked the HuR levels in ELF-EMFs-treated HeLa, ES-2, and DU-145 cells. However, no significant changes in HuR levels have been observed in any of the tested cells (Figure 1C).

To further understand the mechanism by which ELF-EMFs may contribute to tumor suppression, we evaluated the levels of tumor necrosis factor alpha (TNF-α) in ELF-EMF-treated cells. TNF-α is a cytokine produced in response to injury, endotoxin exposure, or infection and can paradoxically induce programmed cell death [51,52]. Interestingly, TNF-α was significantly upregulated only in ES-2 cells exposed to ELF-EMFs for 15 and 30 min (* p < 0.05, ** p < 0.01, Figure 1C). Conversely, DU-145 cells were characterized by a diminution of the levels of TNF-α (** p < 0.01, *** p < 0.001, Figure 1C), while no significant changes in the TNF-α secretion were observed in HeLa. Moreover, Sołek et al. have revealed that ELF-EMFs treatment led to down-regulation of the levels of TNF-α in HeLa and DU-145 cells; however, they used different exposure times [42]. Interestingly, TNF-α can also induce ROS production, which has been reported to contribute to mitochondrial and nuclear oxidative DNA damage in cardiac myocytes [53], L929 cell fibroblasts [54], and primary murine hepatocytes [55]. This may suggest that elevated levels of TNF-α in ES-2 cells stimulate ROS levels, which, in consequence, lead to cell death.

Vimentin is a cytoskeleton protein that plays a significant role in cancer development and progression. It has been found to be overexpressed in several cancer types, including prostate and cervical cancer. Its overexpression was correlated with increased tumor growth, invasion, and metastasis. Therefore, we investigated its levels in response to ELF-EMF-treated gynecological and urological cancer cells. However, statistically significant upregulation of VIM levels has been observed in ES-2 cells treated for 15 min and in DU-145 cells treated for 30 min (* p < 0.05, ** p < 0.01, Figure 1C). Interestingly, VIM has been implicated in the generation of ROS in macrophages, thereby contributing to bacterial killing [56].

Hydroxyl ions generated by the Fenton reaction are highly reactive toward DNA [57]. Additionally, our previous studies have shown that short-term exposure to ELF-EMFs (50 Hz, 1.3 mT) modulates the levels of DNA damage and response factors in ARPE-19 and RGC-5 cells [13]. Thus, we checked the expression of different DDR factors at the mRNA or protein levels in HeLa, ES-2, and DU-145 cells (Figure 2A,B). Out of 6 tested genes (H2AFX, OGG1, XRCC5, XRCC6, POLβ, and MAP1LC3B), only the levels of XRCC5 have been significantly upregulated in ES-2 cells treated for 15 and 30 min with ELF-EMFs (Figure 2A). Similar results were obtained by Saine-Jahromy et al., who did not observe significant changes in the mRNA levels of DNA repair factors after 50 Hz ELF-EMFs treatment [58]. Additionally, ELF-EMF treatment led to an increase in the nuclear and cellular fractions of RAD51, APE1, and XRCC1 in ES-2 cells, except for the cellular fraction of APE1 and XRCC1 in cells exposed for 15 min (Figure 2B). On the contrary, XRCC1 cellular fraction was upregulated in HeLa cells treated for 15 min, and RAD51 cellular and nuclear fractions were downregulated in DU-145 cells subjected to 15 and 30 min of ELF-EMFs (Figure 2B).

A m^5^C methyltransferase—NSUN2 and m^6^A methyltransferase—METTL3 are upregulated in cancer cells, including bladder, prostate, kidney, cervical, ovarian, liver, breast, etc. [59,60]. Additionally, several data suggest that NSUN2 and METTL3 play key roles in biological processes, such as proliferation, differentiation, migration, and tumorigenesis in an m^5^C- and m^6^A-dependent manner [61,62]. Interestingly, only ES-2 treated with ELF-EMFs for 15 and 30 min exhibited a significant increase in NSUN2 levels compared to untreated CTR. HeLa cells exhibited significant upregulation of NSUN-2 levels after 15 min of ELF-EMF treatment. (Figure 3A). In the case of METTL3, only ELF-EMF-treated DU-145 cells presented significantly higher levels of the tested protein after 30 min of treatment compared to CTR (Figure 3A). However, further research is needed to fully understand the interplay between ELF-EMFs and RNA methylation and to explore the full potential of this interaction in gynecological and urological cancer cells.

4. Materials and Methods

4.1. Cell Culture and ELF-EMF Treatment

The research was conducted on in vitro cultures of 3 cell lines: HeLa, ES-2, and DU-154, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in the DMEM media (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% (v/v) fetal bovine serum (EUR_X_, Gdansk, Poland), 1% penicillin-streptomycin, and 1% L-glutamine (Corning, Camelback Rd., Glendale, CA, USA) in a humidified atmosphere, 5% CO_2_ at 37 °C. The tested cell lines were studied under a sinusoidal, continuous 50 Hz electromagnetic field, with a magnetic induction of 1.3 mT for 15 and 30 min. Untreated cells served as a negative control. The experimental conditions were selected based on our previous studies [13]. The source of the electromagnetic field was controlled by an innovative generator installed inside the incubator Cell Expert C170 (Eppendorf, Hamburg, Germany). Parameters required for the cells’ growth were provided by the cell culture incubator.

4.2. Reactive Oxygen Species (ROS) Levels Analysis

The reactive oxygen species detection was performed using dihydroethidium (DHE, #D11347, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were seeded at a density of 5 × 10^4^ onto 96-well plates and exposed to the ELF-EMFs as previously described. Next, the DHE probe was added at a dilution of 1:1000 and incubated for 30 min in a humidified atmosphere, 5% CO_2_ at 37 °C. After the incubation time, the media was exchanged, and analyses were performed at time 0 h (right after the media exchange) and 24 h after the media with the probe was removed. The fluorescence measurement was performed using the GloMax^®^ Discover Microplate Reader (Promega GmbH, Walldorf, Germany).

4.3. Immunofluorescence

HeLa, ES-2, and DU-145 cells were treated with 50 Hz sinusoidal ELF-EMF for 15 min and 30 min and incubated for 24 h in a humidified atmosphere, 5% CO_2_ at 37 °C. Next, cells were fixed and subjected to an immunostaining protocol as previously described [13]. The cells were incubated with primary antibodies: BCL-2 (1:50, SAB5700155), NSUN2 (1:100, BT-AP11581), METTL3 (1:50, RA20420), APE1 (1:100, BT-AP07734), RAD51 (1:100, PA5-27195), HuR (1:100, #07-1735), HO-2 (1:100, PA5-28334), XRCC1 (1:50, MA5-13412), VIM (1:250, MA5-11883), HSP90 (1:100, MA1-10372), and TNF-α (1:100, #60291-1-IG) (Thermo Fisher Scientific, Waltham, MA, USA or Merck, Schnelldorf, Germany) at 4 °C overnight. Next, the secondary antibodies conjugated to Texas Red (1:1000, T2767) or FITC (1:1000, F2761) (Thermo Fisher Scientific, Waltham, MA, USA) were applied at room temperature for 1 h. Nuclei were stained using Hoechst 33342. Cell images were acquired using an IN Cell Analyzer 2000 confocal imaging system and analyzed using IN Cell Analyzer 1000 Software (GE Healthcare Life Sciences, Piscataway, NJ, USA). The immunofluorescent signals of the protein levels are presented as relative fluorescent units (RFUs). IN Cell Analyzer 1000 Software uses image segmentation algorithms to separate different cellular compartments. The program identifies nuclei by detecting fluorescence from a nuclear stain (e.g., Hoechst 33342). The region between the nuclear boundary and the whole-cell boundary is assigned as the cytoplasm. Quantitative measurements (such as intensity of a protein signal) are then extracted separately for the nuclear and cytoplasmic compartments. This allows accurate assessment of protein localization and distribution within cells without the need for biochemical fractionation.

4.4. Real-Time Polymerase Chain Reaction (RT-PCR)

For the RNA extraction, cells were cultured and treated with ELF-EMFs as described in Section 4.1. Total RNA was isolated from HeLa and ES-2 cells with the use of the Total RNA Maxi kit (A&A Biotechnology, Gdansk, Poland) and subsequently used for cDNA synthesis with High-Capacity cDNA Reverse Transcription Kit (cat. no. 4374966, Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocols. Next, the Real-time PCR (RT-PCR) method, using the Applied Biosystems StepOnePlus™ Real-Time PCR System, was performed to determine the expression of the investigated genes. The following TaqMan^®^ Gene Expression Assays (Life Technologies Corporation, Carlsbad, CA, USA) were used: NFE2L2 (assay ID: Hs00975961_g1), MAP1LC3B (assay ID: Hs00797944_s1), H2AFX (assay ID: Hs00266783_s1), OGG1 (Hs00213454_m1), XRCC6 (assay ID: Hs01922655_g1), XRCC5 (assay ID: Hs00897854_m1), and POLβ (assay ID: Hs01099715_m1). Relative gene expression was normalized using the delta-delta Ct method with GAPDH (assay ID: Hs99999905_m1) as the reference gene.

4.5. Statistical Analysis

All experiments were performed with a minimum of three independent repetitions, while RT-PCR experiments were carried out with two independent replicates. The results represent the mean ± SD. Differences between the control and treated samples were analyzed using a one-way ANOVA and Dunnett’s test, whereas differences between samples were calculated using a one-way ANOVA and Tukey’s multiple comparison test. Statistical significance was assessed using GraphPad Prism 8. p-values of less than 0.05 were considered significant.

5. Conclusions

We have investigated, for the first time, the effect of short-term exposure of gynecological (HeLa and ES-2) and urological (DU-145) cells to t50 Hz ELF-EMFs with a magnetic flux 1.3 mT (Figure 4). We showed that exposure to ELF-EMFs for 30 min induces elevated ROS production and changes in the levels of DDR and epigenetic modification factors in HeLa, ES-2, and DU-145 cells. The strongest effect of ELF-EMF treatment was documented in ES-2 cells, where increased ROS levels were accompanied by significant upregulation of XRCC5 gene expression and elevated levels of several proteins: TNF-α, RAD51, APE1, XRCC1, and NSUN2. In contrast, DU-145 cells displayed decreased levels of BCL-2, HSP90, RAD51, and TNF-α, along with increased expression of VIM and METTL3, in response to 30 min of ELF-EMF treatment, while HeLa cells showed diminished levels of HSP90. Overall, we suggest that already short-term treatment with a 50 Hz ELF-EMF may serve as a promising treatment strategy against urological and gynecological cancer.