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Pulsed Electromagnetic Field Enhances Doxorubicin-induced Reduction in the Viability of MCF-7 Breast Cancer Cells
Korean J Clin Lab Sci 2024;56:73-84  
Published on March 31, 2024
Copyright © 2024 Korean Society for Clinical Laboratory Science.

Sung-Hun WOO and Yoon Suk KIM

Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, Wonju, Korea
Correspondence to: Yoon Suk KIM
Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, 1 Yeonsedae-gil, Heungeop-myeon, Wonju 26493, Korea
E-mail: yoonsukkim@yonsei.ac.kr
ORCID: https://orcid.org/0000-0002-1956-0126
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
A pulsed electromagnetic field (PEMF) enhances the efficacy of several anticancer drugs. Doxorubicin (DOX) is an anticancer agent used to treat various malignancies, including breast cancer. This study examined whether a PEMF increases the anticancer effect of DOX on MCF-7 human breast cancer cells and elucidated the underlying mechanisms affected by PEMF stimulation in DOX-treated MCF-7 human breast cancer cells. A cotreatment with DOX and a PEMF potentiated the reduction in MCF-7 cell viability compared to the treatment with DOX alone. The PEMF elevated DOX-induced G1 arrest by affecting cyclin-dependent kinase 2 phosphorylation and the expression of G1 arrest-related molecules, including p53, p21, cyclin E2, and polo like kinase 1. In addition, PEMF increased the DOX-induced upregulation of proapoptotic proteins, such as Fas and Bcl-2-associated X, and the downregulation of antiapoptotic proteins, including myeloid leukemia 1 and survivin. PEMF promoted the DOX-induced activation of caspases-8, -9, and -7 and poly (adenosine diphosphateribose) polymerase cleavage in MCF-7 cells. In conclusion, PEMF enhances the anticancer activity in DOX-treated MCF-7 breast cancer cells by increasing G1 cell cycle arrest and caspase-dependent apoptosis. These findings highlight the potential use of a PEMF as an adjuvant treatment for DOX-based chemotherapy against breast cancer.
Keywords : Apoptosis, Breast neoplasm, Cell cycle checkpoints, Doxorubicin, Pulsed electromagnetic field
INTRODUCTION

Breast cancer frequently occurs among women with high mortality rates worldwide, and chemotherapy has been used as a standard treatment for it [1]. However, long-term chemotherapy leads to drug resistance, leaving few effective treatment options available [1]. In addition to chemoresistance, women treated with chemotherapy experience numerous side effects from excessive concentrations of the drug [2]. Recently, various strategies combined with chemotherapy have been developed to overcome these problems. One possible approach is pulsed electromagnetic field (PEMF). PEMF is one of the complementary and alternative medicines and has been approved by the American Food and Drug Administration [3]. PEMF, a technique where a coil powered by fluctuating electric currents is used, generates dynamic magnetic fields which have been suggested to elicit cellular responses by directly inducing electrical currents in the biological tissue. Key benefits of PEMF include its high acceptance due to minimal side effects, non-invasive approach, and straightforward therapeutic application [4]. Currently, PEMF has been mainly used as an adjunct therapy to bone repair and pain alleviation. PEMF is also known to influence the recovery of neurological diseases such as Parkin’s disease, Alzheimer’s disease, or multiple sclerosis [5]. In addition, PEMF is developing as a novel treatment for the regulation of inflammation [6]. Several studies have also demonstrated that PEMF can serve as adjuvant to improve the anticancer effects of chemotherapeutic drugs on breast cancer cells. PEMF can promote proapoptotic effect of pemetrexed on MCF-7 breast cancer cells [7]. In addition, EMF increases the cytotoxic effect of doxorubicin (DOX) on HBL-100 breast cancer cells [8]. PEMF also induced a significant increase in the apoptosis of etoposide-treated MCF-7 breast cancer cells [9]. Recently, there was a study reporting that treatment with extremely low frequency (ELF)-EMF can increase the cytotoxic effect of DOX on MCF-7 breast cancer cells compared with treatment with DOX alone [10].

DOX is one of the effective anticancer drugs used to treat breast cancer. DOX exerts anticancer effects through various mechanisms. It can lead to cell cycle arrest in G1 phase, resulting in suppression of cell growth and induction of cell death [11]. G1 arrest is regulated by coordinated interactions between cell cycle-related proteins such as p53. When a cell detects abnormal conditions including DNA damage, p53 is activated and induces upregulation of various proteins including p21 and p27. p21 and p27 suppresses the activity of cyclin-dependent kinase 2 (CDK2) which plays a major role in transition from G1 to S phase, ultimately halting the progression of the cell cycle in G1 phase [11, 12]. DOX caused G1 arrest by increasing p53 expression, thereby inhibiting the growth of SH-SY5Y neuroblastoma cells [13]. DOX also suppressed the proliferation of MCF-7 breast cancer cells by inducing G1 arrest, which is mediated by upregulation of p21 [14]. In addition, treatment with DOX resulted in G1 cell cycle arrest by inducing expression of p53 and p21 in HepG2 hepatoma cells [15]. In A549 human lung cancer cells, DOX affected the expression of p27 and triggers cell cycle arrest [16]. Furthermore, DOX suppressed the expression of CDK2 gene and enhanced G1 arrest in curcumin-treated gastric and prostate cancer cells [17].

Until now, the underlying mechanisms by which PEMF affects the DOX-induced reduction in the viability of MCF-7 breast cancer cells are not fully understood. In this study, we report here that PEMF enhances DOX-induced cell cycle arrest in G1 phase and caspase-dependent apoptosis, consequently leading to a further reduction in the viability in DOX-treated MCF-7 cells. According to our findings, we suggest that PEMF could be applied as a potential adjuvant treatment for DOX-based chemotherapy against breast cancer.

MATERIALS AND METHODS

1. Materials

DOX was purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-ethylenediaminetetraacetic acid (EDTA), and trypan blue solution were all obtained from Thermo Fisher Scientific Inc. Propidium iodide (2.5 mg/mL) was purchased from BD Biosciences. We purchased antibodies against p53, p21, p27, cyclin E2, polo like kinase 1 (Plk1), Bcl-2-associated X (Bax), Fas, t-Bid, apoptosis inducing factor (AIF), survivin, X-linked inhibitor of apoptosis (XIAP), B-cell lymphoma 2 (Bcl-2), myeloid leukemia 1 (Mcl-1), cleaved caspase-9, cleaved caspase-8, cleaved caspase-7, cleaved caspase-3, poly (adenosine diphosphate-ribose) polymerase (PARP), and cleaved PARP from Cell Signaling Technology Inc. Antibody against β-actin was purchased from Santa Cruz Biotechnology.

2. Cell Culture and PEMF Stimulation

The human breast adenocarcinoma cell line MCF-7 (American Type Culture Collection) was cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin. It was maintained at 37℃ in a humidified atmosphere with 5% CO2. For PEMF stimulation, cells were seeded at a density of 1×105 cells into each well of a 6-well plate. After 24 hours, cells were treated with DOX and stimulated with or without a 1 hour long PEMF session (2.5 mT at 70 Hz as repetitive biphasic pulses) thrice (with interval of 4 hours) a day for 3 days.

3. Cell Viability Assay

To assess cell viability by using trypan blue exclusion assay, the cultured medium was removed, and 0.25% trypsin-EDTA (0.5 mL per well) was applied to the cells for 5 minutes. Afterward, the cells were neutralized with 1.5 mL of 10% DMEM. The cells were detached, and an equal volume of suspended cells was mixed with 0.4% trypan blue dye solution (Thermo Fisher Scientific Inc.). The number of viable cells was enumerated by a hemocytometer (Paul Marienfeld GmbH & Co. KG).

To analyze cell viability by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Ez-cytox assay kit (iTSBiO) was used. In details, MDA-MB-231 cells at the density of 5×104 were seeded in 96-well plates and treated with 0.05 μM of DOX in the absence and presence of PEMF stimulation for 3 days. After then, ten microliters of water soluble tetrazolium salts reagent was added to each well and incubated at 37℃ with 5% CO2 for 1 hour. The cell viability was measured at 450 nm using NanoQuant Infinite M200 (Tecan).

4. Cell Cycle Analysis

Trypsinized cells were washed twice with PBS, fixed with 70% ethanol in PBS and incubated for 2 hours at 4℃. The fixed cells were stained with a solution containing ribonuclease A (0.1 mg/mL) and propidium iodide (2.5 mg/mL) in PBS. After incubation for 30 minutes at 37℃, the cells were rinsed, and the DNA content was assessed using FACSCalibur (BD Biosciences). At least 10,000 cells per sample were analyzed by using the CellQuestPro software program (BD Biosciences).

5. Western Blot Analysis

MCF-7 cells were washed with PBS and lysed at 4℃ in PBS containing 1% Triton X-100, protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail (Roche Holding AG). The supernatant was collected after centrifugation at 19,000×g for 10 minutes at 4℃, and protein concentration was determined using the Lowry protein assay (Bio-Rad Laboratories Inc.). Protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 120 minutes. Then, proteins were transferred onto a nitrocellulose membrane (Pall Corp.) for 2 hours. Protein blots were blocked with 5% (w/v) skim milk (BD Biosciences) for 30 minutes and then incubated overnight with primary antibody at 4℃. Each immunoblot was incubated with the appropriate horseradish peroxidase-labeled secondary antibody (anti-mouse or anti-rabbit). The immune-labeled proteins were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific Inc.). The band intensities of each protein were quantified and normalized relative to β-actin band intensities with ImageJ (National Institutes of Health).

6. Statistical Analysis

Quantified data were statistically evaluated by Student’s t-test using GraphPad Prism 8 (GraphPad Software Inc.). All experiments were conducted at least thrice. The values are depicted using the mean and standard error of the mean (SEM). Differences between the means of the groups were considered to be statistically significant if P<0.05, P<0.01, and P<0.001.

RESULTS

1. PEMF Promotes DOX-induced Decrease in Cell Viability in MCF-7 Breast Cancer Cells

Several reports have demonstrated that PEMF improves the anticancer effect of certain chemotherapeutic drugs. Therefore, we first examined whether PEMF affects the anticancer efficacy of DOX on MCF-7 breast cancer cells. MCF-7 cells were treated with 0.05 μM of DOX and stimulated with or without PEMF for 3 days. Then, the viable cells were enumerated using trypan blue dye exclusion assay. Cotreatment with DOX and PEMF further decreased the viable cells when compared to treatment with DOX alone (Figure 1A). Next, to investigate the kinetics of the reduction in cell viability in MCF-7 cells treated with DOX and PEMF, cells were treated with DOX in the absence or presence of PEMF for the indicated periods (1, 2, or 3 days) and viable cells were then counted. Compared to DOX-treated cells, there were no significant changes in cell viability in cells treated with DOX and PEMF on the first day after treatment. However, the viable cells had significantly reduced from the second day after the treatment onward (Figure 1B). These data show that PEMF enhances the reduction in cell viability in DOX-treated MCF-7 cells in a time-dependent manner.

Fig. 1. Enhancement of DOX-induced decrease of MCF-7 cell viability in response to PEMF stimulations. MCF-7 cells were treated with 0.05 μM DOX followed by incubation for 3 days. During this period, the treated cells were stimulated with or without a 1 hours PEMF session (2.5 mT at 70 Hz) thrice a day for three consecutive days. The viable cells were then measured by (A) trypan blue dye exclusion assay and (B) EZ-cytox assay. (C) MCF-7 cells were treated with 0.05 μM DOX and then were left unstimulated or stimulated with PEMF for the indicated periods. Then, the viable cells were enumerated by trypan blue dye exclusion assay. The number of viable cells in the Con was set as 100%. All data were expressed as the mean±standard error of the mean of three independent experiments. P-values were determined using Student’s t-tests (*P<0.05, **P<0.01, and ***P<0.001).
Abbreviations: DOX, doxorubicin; PEMF, pulsed electromagnetic field; Con, control group; ns, not significant; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

2. PEMF Enhances DOX-induced G1 Arrest in MCF-7 Cells

DOX exerts antiproliferative effect on a wide range of cancer cells by inhibiting cell cycle. Therefore, we investigated whether PEMF affects cell cycle in DOX-treated MCF-7 cells. Cells were treated with DOX in the absence or presence of PEMF stimulations for 3 days and the cell cycle analysis was performed using flow cytometry. Cotreatment with DOX and PEMF elevated the number of MCF-7 cells arrested in the G1 phase in a time-dependent manner when compared to DOX treatment alone (Figure 2). There were no significant differences in cell cycle between untreated control cells and cells stimulated with PEMF only. Our data show that PEMF increases DOX-induced cell cycle arrest in the G1 phase of MCF-7 cells.

Fig. 2. PEMF-potentiated cell cycle arrest in G1 phase in DOX-treated MCF-7 cells. MCF-7 cells were treated with 0.05 μM DOX in the absence or presence of PEMF stimulation for the indicated periods. (A) Cells were harvested and stained with propidium iodide at the indicated times. Then, the cell cycle was analyzed by flow cytometry. (B) The percentage of the G1 population is displayed as a bar graph. Data were represented as the mean±standard error of the mean of three independent experiments. P-values were determined using the Student’s t-test (*P<0.05 and **P<0.01).
Abbreviations: PEMF, pulsed electromagnetic field; DOX, doxorubicin; ns, not significant; Con, control group; FL2-A, fluorescence channel 2-area.

3. PEMF Influences the Expression of G1 Arrest-related Molecules in DOX-treated MCF-7 Cells

Several molecules are involved as negative or positive regulators in cell cycle arrest in G1 phase. Thus, we examined whether PEMF stimulation affects expression of G1 arrest-related molecules. MCF-7 cells were treated with DOX in the absence or presence of PEMF for 48 hours and then harvested for western blot assay. The expression of p53, which inhibits transition from G1 to S phase in cell cycle, was higher in DOX/PEMF-treated cells than in DOX-treated cells (Figure 3A). Next, we examined expression p21 and p27, which are regulated by p53 and involve in G1 arrest by binding to and inhibiting proteins such as CDK2-cyclin E2 complex. The expression of p21 was further increased in the DOX/PEMF-treated cells than in DOX-treated cells (Figure 3B). However, no significant differences in the expression of p27 were observed between cells treated with DOX only and cells treated with DOX and PEMF. Furthermore, the combined treatment with DOX and PEMF produced the further reduction in CDK2 phosphorylation and cyclin E2 expression when compared to treatment with DOX alone (Figure 3C). In addition, since there was a study reporting that degradation of Plk1 is associated with cell cycle arrest in G1 phase, expression of Plk1 was also assessed. Plk1 expression was decreased in the DOX-treated cells and further reduced in the DOX/PEMF-treated cells (Figure 3D). Our results show that PEMF affects the phosphorylation of CDK2 and the expression of p53, p21, cyclin E2 and Plk1, implying that PEMF enhances DOX-induced G1 arrest by influencing these molecules.

Fig. 3. PEMF-enhanced regulation of G1 arrest-related molecules in DOX-treated MCF-7 cells. MCF-7 cells were treated with 0.05 μM DOX. Then, the cells were stimulated with PEMF for 2 days. (A) p53, (B) p21 and p27, (C) p-CDK2 and cyclin E2, and (D) Plk1 were assessed using western blot assay. β-actin was used as an internal control. Densitometry analysis of each protein band was performed using ImageJ program and normalized by β-actin level. The level of proteins in the Con was set as 1. Data are shown as the mean±standard error of the mean. P-values were determined using Student’s t-tests (*P<0.05 and ***P<0.001).
Abbreviations: PEMF, pulsed electromagnetic field; DOX, doxorubicin; p-CDK2, phosphorylated cyclin-dependent kinase 2; Plk1, polo like kinase 1; Con, control group; ns, not significant.

4. PEMF Enhances the Caspase-dependent Apoptosis in DOX-treated MCF-7 Cells

DOX also exerts anticancer effect by inducing apoptosis and sustained arrest of cell cycle is one of triggers for apoptotic cell death. Therefore, we investigated whether PEMF potentiates DOX-induced cell death in MCF-7 cells. The cells were treated with DOX, stimulated with PEMF for 3 days, and then harvested for western blot assay. First, cleaved PARP, a well-known marker for apoptosis; was observed. The DOX-treated cells exhibited increased PARP cleavage compared with the control and further increased PARP cleavage was observed in the DOX/PEMF-treated cells (Figure 4A). Next, the activation of executioner caspases (caspase-3 and -7) was examined. Cleaved caspase-7 was higher in the DOX-treated cells than in the control group and was further higher in the DOX/PEMF-treated cells (Figure 4B). However, no significant differences in caspase-3 cleavage were found between the DOX-treated cells and DOX/PEMF-treated cells. We also investigated the activation of initiator caspases (caspase-8 and -9). Cleavage of caspase-8 and -9 were elevated in the DOX-treated cells and increased even more in the DOX/PEMF-treated cells (Figure 4B). These results show that PEMF enhances caspase-dependent apoptosis pathway in DOX-treated MCF-7 cells. In addition, to examined whether PEMF affects apoptosis-related molecules in DOX-treated MCF-7 cells, we assessed the expression levels of proapoptotic molecules including Fas, t-Bid, Bax and AIF, and antiapoptotic molecules including Bcl-2, Mcl-1, survivin and XIAP. The expression of Fas and Bax was elevated to a larger degree in the DOX/PEMF-treated cells than in the DOX-treated cells (Figure 4C) and the expression of survivin was decreased in the DOX-treated cells and further reduced in the DOX/PEMF-treated cells (Figure 4D). In addition, Mcl-1 expression was reduced in the DOX/PEMF-treated cells compared to the DOX-treated cells (Figure 4D). However, there were no significant differences in the truncation of Bid and expression of AIF, Bcl-2 and XIAP between the DOX-treated cells and DOX/PEMF-treated cells (data not shown). These findings show that PEMF influences apoptosis-related molecules such as Fas, Bax, Mcl-1, and survivin, activates apoptosis-related caspases such as caspase-9/8/7, implying that PEMF enhances DOX-stimulated caspase-dependent apoptotic cell death by regulating the expression of Fas, Bax, Mcl-1, and survivin.

Fig. 4. Increase in DOX-induced apoptotic cell death in response to PEMF stimulations. MCF-7 cells were treated with 0.05 μM of DOX and stimulated with PEMF for 3 days. After that, (A) cleavage of PARP, (B) activation of apoptotic caspases, (C) protein levels of proapoptotic molecules, and (D) amounts of antiapoptotic molecules were analyzed using western blot assay. β-actin was used as an internal control. Densitometry analysis of each protein band was performed using ImageJ program and normalized by β-actin level. The level of proteins in the Con was set as 1. Data are shown as the mean±standard error of the mean. P-values were determined using Student’s t-tests (*P<0.05, **P<0.01, and ***P<0.001).
Abbreviations: DOX, doxorubicin; PEMF, pulsed electromagnetic field; PARP, poly (adenosine diphosphate-ribose) polymerase; Bax, Bcl-2-associated X; Mcl-1, myeloid leukemia 1; Con, control group; ns, not significant.
DISCUSSION

While PEMF has been suggested as an adjuvant method for anticancer chemotherapy, the potency of PEMF on DOX-treated breast cancer cells and the mechanisms underlying its effect have not been fully understood. We found here that PEMF (i) increases the reduction in the cell viability in DOX-treated MCF-7 cells; (ii) regulates a variety of cell cycle-related molecules and enhances DOX-induced cell cycle arrest in G1 phase; (iii) influences the expression of apoptosis-related molecules and promotes DOX-induced caspase-dependent apoptosis (Figure 5).

Fig. 5. Schematic diagram of PEMF-enhanced G1 arrest and apoptosis in DOX-treated MCF-7 cells.
Abbreviations: PEMF, pulsed electromagnetic field; DOX, doxorubicin; Plk1, polo like kinase 1; CDK2, cyclin-dependent kinase 2; Mcl-1, myeloid leukemia 1; Bax, Bcl-2-associated X; PARP, poly (adenosine diphosphate-ribose) polymerase.

PEMF generates a low-frequency magnetic field with specified waveforms and amplitudes ranging from 6 to 500 Hz that produces bioelectric currents in biological tissues, inducing special therapeutic effects [18]. However, there are no established PEMF parameters for anticancer therapy. In our PEMF system, treating cells with PEMF at 70 Hz and 2.5 mT showed no change in cell viability (Figure 1A). A recent paper has also shown that ELF-EMF at 50 Hz and 20 mT did not affect cell viability of MCF-7 cells [10] and the work of Loja et al [19] demonstrated that PEMF (15 Hz, 5 mT) did not change the growth of acute lymphoblastic leukemia and lymphoma cell lines. On the other hand, a previous study has revealed that cell viability of MCF-7 cells is impaired when exposed to PEMF at frequencies of 20~50 Hz and amplitudes of 2~5 mT [20]. In addition, exposure to a magnetic field with an amplitude of 35 mT and a frequency of 50 Hz induced apoptosis in human lymphocytic leukemia cells [21]. de Seze et al [22] also reported that PEMF (0.18 T, 0.8 Hz) inhibited proliferation in cervical cancer cells. These controversial findings may be due to differences in the PEMF devices used, duration, and cell types. Therefore, further research is needed to optimize the parameters of PEMF for applying cancer treatment.

The frequency of electromagnetic fields used in clinical treatment is usually less than 100 Hz and the magnetic flux density is between 0.1 mT to 30 mT [23]. We used PEMF stimulation with a frequency of 70 Hz and an intensity of 2.5 mT, suggesting that the frequency and intensity of PEMF used in this study are appropriate for clinical applicability. Furthermore, we showed that PEMF increases the effect of DOX in MCF-7 cells, implying that the lower dose of DOX can be used for treatment of breast cancer by using PEMF as an adjuvant procedure. PEMF appears to have potential as an adjuvant strategy in clinical settings to reduce the side effects and drug resistance that can occur when patients are treated with high concentrations of DOX.

Recent research has observed that exposure to ELF-EMF can promote the growth-inhibitory properties of DOX and DOX-induced G1 arrest in MCF-7 breast cancer cells [10], but has not fully clarified the mechanism by which PEMF contributes to DOX-induced G1 arrest. In this study, we observed that the expression of p53 and p21 was increased in DOX/PEMF-treated cells compared to DOX-treated cells (Figure 3). p53 is known to be a key player causing G1 arrest and regulates p21 expression. p21 is also involved in G1 arrest by binding to and inhibiting CDK2-cyclin E2 complex [12]. These findings imply that PEMF may influence p53, causing increase of p21 expression, and upregulated p21 subsequently suppresses CDK2-cyclin E2 complex, which is major complex implicated in transition from G1 to S phase [12], leading to G1 arrest. In addition, we also found that phosphorylation of CDK2 and expression of cyclin E2 were decreased in the DOX/PEMF-treated cells when compared with those in DOX-treated cells (Figure 3). These suggest that PEMF may cause the reduction of CDK2 phosphorylation and the increase of cyclin E2 degradation, which in turn lead to the reduction in the activity of CDK2-cyclin E2 complex, consequently enhancing G1 arrest in DOX-treated MCF-7 cells. The aforementioned study showed that ELF-EMF induces reactive oxygen species (ROS) accumulation and suggested that ROS may be involved in ELF-EMF-induced increase of G1 arrest in DOX-treated MCF-7 cells [10]. There is a study reporting that G1 arrest is mediated via ROS/ataxia telangiectasia mutated (ATM)/p53 pathway in A549 lung epithelial cancer cells [24]. In addition, another report showed that ROS induces G1 arrest in lung cancer cells via the activation of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinases (JNK) pathway, which lead to upregulation of c-Jun, p53, p21, and p27 [25]. It is possible that ROS may be an upstream molecule of p53 in PEMF-enhanced G1 arrest in DOX-treated MCF-7 cells. It seems to be needed to determine whether PEMF influences ATM and/or ASK1/JNK pathway in DOX-treated MCF-7 cells. Meanwhile, Plk1 was known to function mainly in the late G2 and mitotic phases [26]. Recently, there has been an increasing number of studies reporting that Plk1 also plays a role in the G1 and S phases [27]. Inhibition of Plk1 increased gemcitabine-induced G1 arrest in human pancreatic cancer cells [28]. Giráldez et al [27] also showed that the degradation of Plk1 induced arrests of G1 cell cycle in HeLa cervical cancer cells. We found that DOX weakened Plk1 expression in MCF-7 cells and PEMF further reduced the DOX-induced reduction in Plk1 expression (Figure 3), suggesting that PEMF may promote DOX-induced G1 arrest by downregulating the expression of Plk1.

A Recent reports proposed that ELF-EMF enhances DOX-induced apoptosis in MCF-7 cells [10]. However, the underlying molecular mechanisms have not fully determined. In this study, we found that PEMF promotes DOX-induced cleavage of PARP and activation of caspase-7/8/9 was increased (Figure 4). In addition, PEMF enhanced the expression of Fas and Bax and suppressed the expression of survivin and Mcl-1. Our findings suggest that PEMF (i) upregulates Fas, which in turn activates caspase-8-dependent extrinsic apoptotic pathway; (ii) enhances upregulation of Bax and downregulation of Mcl-1, which subsequently activates caspase-9-dependent intrinsic apoptotic pathway; (iii) downregulates survivin, an inhibitor of executioner caspases such as caspase-3 and -7, ultimately resulting in enhancement of apoptosis. p53 is known to regulate the expression of several apoptosis-related molecules. p53 increased the expression of the proapoptotic genes including Fas, Bax, Bcl-2 interacting mediator of cell death, p53 upregulated modulator of apoptosis (PUMA), NOXA and Bcl-2 homologous antagonist killer. Also, p53 regulated apoptosis-related molecules such as Bcl-2 family, survivin, Mcl-1, PUMA, NOXA, and Bid [29]. Therefore, p53 could be an important molecule to regulate the expression of Fas, Bax, survivin, and Mcl-1 in DOX/PEMF-treated MCF-7 cells. Furthermore, it has been reported that Plk1 depletion triggers cell death on several types of cancer by inducing apoptosis [30], implying that Plk1 may be involved in apoptosis as well as cell cycle arrest in DOX/PEMF-treated MCF-7 cells.

In conclusion, we report that PEMF stimulation enhances the reduction in the cell viability by enhancing cell cycle arrest and apoptosis in MCF-7 breast cancer cells. Our findings may serve as a basis for using PEMF as an adjuvant treatment in the DOX-based breast cancer treatment.

요 약

펄스 전자기장(pulsed electromagnetic field, PEMF)은 여러 항암제의 항암 효과를 향상시키는 것으로 알려져 있고 독소루비신(doxorubicin, DOX)은 유방암을 포함한 다양한 종류의 악성 종양을 치료하는 데 사용되는 항암제이다. 본 연구는 PEMF가 MCF-7 유방암 세포에 대한 DOX의 항암 효과 증진 여부를 조사하고 관련기전을 규명하기 위해 진행되었다. 본 연구팀은 DOX와 PEMF를 동시에 처리하면 DOX 단독 처리에 비해 MCF-7 유방암 세포의 생존율 감소가 더 커지는 것을 확인하였다. PEMF는 cyclin-dependent kinase 2의 인산화와 p53, p21, 사이클린 E2 및 polo like kinase 1의 단백질 발현에 영향을 주어 DOX 처리에 의한 G1 세포주기 정지를 더욱 증가시켰다. 또한, PEMF는 DOX 처리에 의한 Fas와 Bcl-2-associated X의 증가, myeloid leukemia 1과 survivin의 감소, 카스파제(caspase)-8/9/7의 활성 및 poly (adenosine diphosphate-ribose) polymerase 절단을 더욱 증가시켰다. 이러한 연구결과를 바탕으로, 본 연구팀은 PEMF는 DOX 처리에 의한 G1 세포주기 정지와 카스파제 의존적 세포자멸사를 더욱 증가시켜 DOX 처리에 의한 MCF-7 세포의 생존율 감소를 더욱 증진시킴을 확인할 수 있었다.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (2018R1D1A1B07049134).

Funding

None

Conflict of interest

None

Author’s information (Position)

Woo SH, Graduate student; Kim YS, Professor.

Author Contributions

-Conceptualization: Woo SH, Kim YS.

-Data curation: Woo SH, Kim YS.

-Formal analysis: Woo SH, Kim YS.

-Methodology: Woo SH, Kim YS.

-Software: Woo SH, Kim YS.

-Validation: Woo SH, Kim YS.

-Investigation: Woo SH.

-Writing - original draft: Woo SH, Kim YS.

-Writing - review & editing: Woo SH, Kim YS.

Ethics approval

This article does not require IRB/IACUC approval because there are no human and animal participants.

References
  1. Yang R, Guo Z, Zhao Y, Ma L, Li B, Yang C. Compound 968 reverses adriamycin resistance in breast cancer MCF-7ADR cells via inhibiting P-glycoprotein function independently of glutaminase. Cell Death Discov. 2021;7:204. https://doi.org/10.1038/s41420-021-00590-1.
    Pubmed KoreaMed CrossRef
  2. Gillaizeau F, Chan E, Trinquart L, Colombet I, Walton RT, Rège-Walther MRège-Walther M, et al. Computerized advice on drug dosage to improve prescribing practice. Cochrane Database Syst Rev. 2013;11. https://doi.org/10.1002/14651858.cd002894.pub3.
    Pubmed CrossRef
  3. Dolkart O, Kazum E, Rosenthal Y, Sher O, Morag G, Yakobson EYakobson E, et al. Effects of focused continuous pulsed electromagnetic field therapy on early tendon-to-bone healing. Bone Joint Res. 2021;10:298-306. https://doi.org/10.1302/2046-3758.105.bjr-2020-0253.r2.
    Pubmed KoreaMed CrossRef
  4. Overholt TL, Ross C, Evans RJ, Walker SJ. Pulsed electromagnetic field therapy as a complementary alternative for chronic pelvic pain management in an interstitial cystitis/bladder pain syndrome patient. Case Rep Urol. 2019;2019. https://doi.org/10.1155/2019/5767568.
    Pubmed KoreaMed CrossRef
  5. Funk RHW, Fähnle M. A short review on the influence of magnetic fields on neurological diseases. Front Biosci (Schol Ed). 2021;13:181-189. https://doi.org/10.52586/s561.
    Pubmed CrossRef
  6. Vadalà M, Morales-Medina JC, Vallelunga A, Palmieri B, Laurino C, Iannitti T. Mechanisms and therapeutic effectiveness of pulsed electromagnetic field therapy in oncology. Cancer Med. 2016;5:3128-3139. https://doi.org/10.1002/cam4.861.
    Pubmed KoreaMed CrossRef
  7. Chen MY, Li J, Zhang N, Waldorff EI, Ryaby JT, Fedor PFedor P, et al. In vitro and in vivo study of the effect of osteogenic pulsed electromagnetic fields on breast and lung cancer cells. Technol Cancer Res Treat. 2022;21. https://doi.org/10.1177/15330338221124658.
    Pubmed KoreaMed CrossRef
  8. Sengupta S, Balla VK. A review on the use of magnetic fields and ultrasound for non-invasive cancer treatment. J Adv Res. 2018;14:97-111. https://doi.org/10.1016/j.jare.2018.06.003.
    Pubmed KoreaMed CrossRef
  9. Woo SH, Kim B, Kim SH, Jung BC, Lee Y, Kim YS. Pulsed electromagnetic field potentiates etoposide-induced MCF-7 cell death. BMB Rep. 2022;55:148-153. https://doi.org/10.5483/bmbrep.2022.55.3.119.
    Pubmed KoreaMed CrossRef
  10. Ramazi S, Salimian M, Allahverdi A, Kianamiri S, Abdolmaleki P. Synergistic cytotoxic effects of an extremely low-frequency electromagnetic field with doxorubicin on MCF-7 cell line. Sci Rep. 2023;13:8844. https://doi.org/10.1038/s41598-023-35767-4.
    Pubmed KoreaMed CrossRef
  11. Lüpertz R, Wätjen W, Kahl R, Chovolou Y. Dose- and time-dependent effects of doxorubicin on cytotoxicity, cell cycle and apoptotic cell death in human colon cancer cells. Toxicology. 2010;271:115-121. https://doi.org/10.1016/j.tox.2010.03.012.
    Pubmed CrossRef
  12. Smirnikhina SA, Zaynitdinova MI, Sergeeva VA, Lavrov AV. Improving homology-directed repair in genome editing experiments by influencing the cell cycle. Int J Mol Sci. 2022;23:5992. https://doi.org/10.3390/ijms23115992.
    Pubmed KoreaMed CrossRef
  13. Gogolin S, Ehemann V, Becker G, Brueckner LM, Dreidax D, Bannert SBannert S, et al. CDK4 inhibition restores G(1)-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage. Cell Cycle. 2013;12:1091-1104. https://doi.org/10.4161/cc.24091.
    Pubmed KoreaMed CrossRef
  14. Shao Z, Jiang M, Yu L, Han Q, Shen Z. p53 independent G1 arrest and apoptosis induced by adriamycin. Chin Med Sci J. 1997;12:71-75.
  15. Lee TK, Lau TC, Ng IO. Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother Pharmacol. 2002;49:78-86. https://doi.org/10.1007/s00280-001-0376-4.
    Pubmed CrossRef
  16. Cuadrado M, Gutierrez-Martinez P, Swat A, Nebreda AR, Fernandez-Capetillo O. p27Kip1 stabilization is essential for the maintenance of cell cycle arrest in response to DNA damage. Cancer Res. 2009;69:8726-8732. https://doi.org/10.1158/0008-5472.can-09-0729.
    Pubmed KoreaMed CrossRef
  17. Yaghoubi F, Motlagh NSH, Naghib SM, Haghiralsadat F, Jaliani HZ, Moradi A. A functionalized graphene oxide with improved cytocompatibility for stimuli-responsive co-delivery of curcumin and doxorubicin in cancer treatment. Sci Rep. 2022;12:1959. https://doi.org/10.1038/s41598-022-05793-9.
    Pubmed KoreaMed CrossRef
  18. Waldorff EI, Zhang N, Ryaby JT. Pulsed electromagnetic field applications: a corporate perspective. J Orthop Translat. 2017;9:60-68. https://doi.org/10.1016/j.jot.2017.02.006.
    Pubmed KoreaMed CrossRef
  19. Loja T, Stehlikova O, Palko L, Vrba K, Rampl I, Klabusay M. Influence of pulsed electromagnetic and pulsed vector magnetic potential field on the growth of tumor cells. Electromagn Biol Med. 2014;33:190-197. https://doi.org/10.3109/15368378.2013.800104.
    Pubmed CrossRef
  20. Crocetti S, Beyer C, Schade G, Egli M, Fröhlich J, Franco-Obregón A. Low intensity and frequency pulsed electromagnetic fields selectively impair breast cancer cell viability. PLoS One. 2013;8. https://doi.org/10.1371/journal.pone.0072944.
    Pubmed KoreaMed CrossRef
  21. Radeva M, Berg H. Differences in lethality between cancer cells and human lymphocytes caused by LF-electromagnetic fields. Bioelectromagnetics. 2004;25:503-507. https://doi.org/10.1002/bem.20023.
    Pubmed CrossRef
  22. de Seze R, Tuffet S, Moreau JM, Veyret B. Effects of 100 mT time varying magnetic fields on the growth of tumors in mice. Bioelectromagnetics. 2000;21:107-111. https://doi.org/10.1002/(sici)1521-186x(200002)21:2%3C107::aid-bem5%3E3.0.co;2-6.
    CrossRef
  23. Markov MS. Expanding use of pulsed electromagnetic field therapies. Electromagn Biol Med. 2007;26:257-274. https://doi.org/10.1080/15368370701580806.
    Pubmed CrossRef
  24. Park JS, Park YJ, Kim HR, Chung KH. Polyhexamethylene guanidine phosphate-induced ROS-mediated DNA damage caused cell cycle arrest and apoptosis in lung epithelial cells. J Toxicol Sci. 2019;44:415-424. https://doi.org/10.2131/jts.44.415.
    Pubmed CrossRef
  25. Mhone TG, Chen MC, Kuo CH, Shih TC, Yeh CM, Wang TFWang TF, et al. Daidzein synergizes with gefitinib to induce ROS/JNK/c-Jun activation and inhibit EGFR-STAT/AKT/ERK pathways to enhance lung adenocarcinoma cells chemosensitivity. Int J Biol Sci. 2022;18:3636-3652. https://doi.org/10.7150/ijbs.71870.
    Pubmed KoreaMed CrossRef
  26. Bruinsma W, Raaijmakers JA, Medema RH. Switching Polo-like kinase-1 on and off in time and space. Trends Biochem Sci. 2012;37:534-542. https://doi.org/10.1016/j.tibs.2012.09.005.
    Pubmed CrossRef
  27. Giráldez S, Galindo-Moreno M, Limón-Mortés MC, Rivas AC, Herrero-Ruiz J, Mora-Santos MMora-Santos M, et al. G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis. FASEB J. 2017;31:2925-2936. https://doi.org/10.1096/fj.201601108r.
    Pubmed CrossRef
  28. Li J, Wang R, Schweickert PG, Karki A, Yang Y, Kong YKong Y, et al. Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer. Cell Cycle. 2016;15:711-719. https://doi.org/10.1080/15384101.2016.1148838.
    Pubmed KoreaMed CrossRef
  29. Qian S, Wei Z, Yang W, Huang J, Yang Y, Wang J. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol. 2022;12. https://doi.org/10.3389/fonc.2022.985363.
    Pubmed KoreaMed CrossRef
  30. Liu X, Erikson RL. Polo-like kinase (Plk) 1 depletion induces apoptosis in cancer cells. Proc Natl Acad Sci U S A. 2003;100:5789-5794. https://doi.org/10.1073/pnas.1031523100.
    Pubmed KoreaMed CrossRef

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