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Anticancer and antibacterial potentials induced post short-term exposure to electromagnetic field and silver nanoparticles and related pathological and genetic alterations: in vitro study

Anticancer and antibacterial potentials induced post short-term exposure to electromagnetic field... Background: Resistance to antibiotics and anticancer therapy is a serious global health threat particularly in immu‑ nosuppressed cancer patients. Current study aimed to estimate the antibacterial and anticancer potentials of short‑ term exposure to extremely low frequency electromagnetic field (ELF‑EMF) and silver nanoparticles (AgNPs) either in sole or combined form. Methods: Antibacterial activity was evaluated via determination of the bacterial viable count reduction percentage following exposure, whereas their ability to induce apoptosis in breast cancer (MCF‑7) cell line was detected using annexin V‑fluorescein isothiocyanate and cell cycle analysis. Also, oxidative stress potential and molecular profile were investigated. Results: ELF‑EMF and AgNPs significantly (p < 0.01) reduced K. pneumonia viable count of compared to that of S. aureus in a time dependent manner till reaching 100% inhibition when ELF‑EMF was applied in combination to 10 µM/ml AgNPs for 2 h. Apoptosis induction was obvious following exposure to either ELF‑EMF or AgNPs, however their apoptotic potential was intensified when applied in combination recording significantly (p < 0.001) induced apoptosis as indicated by elevated level of MCF‑7 cells in the Pre G1 phase compared to control. S phase arrest and accumulation of cells in G2/M phase was observed following exposure to AgNPs and EMF, respectively. Up‑regulation in the expression level of p53, iNOS and NF‑kB genes as well as down‑regulation of Bcl‑2 and miRNA‑125b genes were detected post treatment. Conclusions: The antibacterial and anticancer potentials of these agents might be related to their ability to induce oxidative stress, suggesting their potentials as novel candidates for controlling infections and triggering cancer cells towards self‑ destruction. *Correspondence: Shbel.rania@gmail.com Microbiology and Immunology Department, Faculty of Pharmacy, Ahram Canadian University (ACU), 4th Industrial Zone, Banks Complex, 6th October City, Cairo, Egypt Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Infectious Agents and Cancer (2022) 17:4 Page 2 of 16 Keywords: Antibacterial, Anticancer, Electromagnetic field, Silver nanoparticles, Cell cycle, Apoptosis, Oxidative stress Introduction the differential electrical behavior between the cancer The progressing emergence and the rapid spread of bac - and the normal cells. This signaling plays a key role in terial resistance to antibiotics is considered an alarming blocking cellular functions leading to induction of pro- world-wide health problem which strengthens the need grammed cell death [9]. Recently, a study reported that for alternative therapies. This continually growing prob - the tumor suppressive effects of the ELF‐EMF could pre - lem of antibiotic resistance not only endangers the public sent a new approach for the treatment of breast cancer if health, but also endures a massive negative impact on the this technology is clinically applied [10]. It was demon- economic development due to delayed hospitalization strated that the ability of pulsed low-frequency EMF to and recovery time in addition to the need for expensive modify the membrane integrity of cancer cells presents medications as well as specialized care for patients [1]. a new strategy in anticancer therapy. These pulsed mag - Many researchers have directed their efforts to manage netic fields (PMF) could selectively destruct the cancer the problem of antibiotic resistance via estimating the cell membranes without the use of ionizing radiation or effectiveness of new antibacterial agents either alone or cytotoxic agents. Thus, these fields could be applied as in combinations [2]. In the same context, breast cancer is adjuvants in cancer therapy to facilitate the delivery of considered the second common leading cause of cancer anticancer agents to tumor cells [11]. It was also found death among women [3]. Although many cases of cancer that EMF enhanced the in vivo anti-tumor efficacy of cis - initially respond to chemotherapy, but resistance is usu- platin against Lewis lung carcinoma cells [12]. Moreover, ally developed later [4] in addition to the undesirable side the exposure of glioblastoma brain cancer cells to a com- effects that are associated with the currently available bination between EMF and the anticancer agent temozo- chemotherapeutic drugs. Thus, there is also an urgent lomide enhanced the apoptosis via elevated expression demand for developing biocompatible and cost-effective of P53, Bax, and Caspase-3 (pro-apoptotic) genes while anticancer agents [5]. decreasing the expression levels of Bcl-2 and Cyclin-D1 Extremely low frequency electromagnetic field (ELF- (anti-apoptotic) genes [13]. EMF) is one of the most recent applications that exhib- Additionally, the recent advances in nanotechnology ited significant interactions with the living cells. However, offered new horizons in nanomedicine, facilitating the the mechanism of this interaction is still not clarified. synthesis of nanoparticles (NPs) that could be applied as Recent studies were carried out to assess the biological powerful weapon against pathogenic bacteria and cancer influence of such fields on different types of living cells cells [14]. especially on bacterial cells. Multi-directional alterations Among nanotechnology-based therapeutics, AgNPs following bacterial exposure to ELF-EMF were reported attracted the attention of many researchers due to such as ultra-structural and growth kinetics changes their distinctive characteristics and marked therapeutic [6]. Whereas, other studies found that ELF-EMF could potential in treating different diseases [14]. The antimi - enhance or suppress bacterial functional parameters. crobial activities of AgNPs either alone [15] or in com- Therefore, investigating the influence of ELF-EMF on posites with polymer [16] have been demonstrated in bacteria is essential not only for evaluating the impact of addition to their anticancer [17] as well as their antian- environmental stress on biological systems, but also to giogenic potentials [18]. AgNPs are now considered a explore the possibility of using the ELF-EMF to control valuable and non-traditional alternative to antibiotics the resistance to antibiotics [7]. with high antimicrobial potential against multidrug- ELF-EMF generating medical devices were also applied resistant (MDR) Gram-positive and Gram-negative for treatment of cancer patients in intensive care units. bacterial pathogens [19]. It was also reported that Consequently, during the outgoing 25  years research- AgNPs inhibited the proliferation of human glioblas- ers tried to investigate the impact of exposure to ELF- toma cells [20] as well as human breast cancer (MCF- EMF on cellular and molecular behavior in addition to 7) cells [21]. It was also found that AgNPs stimulated its effect on cancer cell metabolism. It was found that pro-apoptotic genes leading to interference with nor- exposure to 50/60  Hz magnetic field  promoted changes mal cellular functions and induction of programmed in signal transduction pathways that were directly associ- cell death. A study reported that AgNPs induced apop- ated to proliferative processes [8]. A study reported that tosis in NIH3T3 fibroblast cells is mediated via gen - electromagnetic field (EMF) could selectively hinder the eration of reactive oxygen species (ROS) and activation oxidation–reduction signaling in cancer cells relying on of Jun N-terminal kinases (JNK) pathway leading to M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 3 of 16 mitochondrial dependent apoptosis [22]. Recently, the Cell culture microenvironmental influence of titanium dioxide NPs Breast cancer (MCF-7) cells (HTB-22) were kindly sup- as a mechanical stimulus on cancer cells has been also plied from the International Center for Training and observed [23]. In addition to the reported concentra- Advanced Researches (ICTAR-Egypt). Cells were cul- tion dependent metabolic disturbing effect of graphene tured in RPMI-1640 media (Hyclone-USA), supple- oxide nanosheets on MCF-7 cells [24]. This alteration mented with 10% heat inactivated fetal bovine serum in the metabolomic profiling of cancer cells could con - (FBS), 100 U/ml penicillin and 100  μg/ml streptomycin. trol many malignant properties that are responsible for Cells were incubated in a humidified atmosphere of 5% tumorigenesis [25]. It was also found that the mecha- CO at 37 °C (Jouan-France) till reaching confluency. nobiological studies of AgNPs in cancer metabolomics suggested that AgNPs might be promising tools that Characterization of commercial AgNPs could be explored to develop enhanced anticancer ther- AgNPs were purchased as a commercial product by apy [26]. Nawah scientific (Cairo-Egypt). Characterization of the Consequently, the present study aimed to evaluate the particle size distribution and surface charge of the pre- antibacterial and the anticancer potentials of short-term pared particles were determined using Zetasizer Nano- exposure to ELF-EMF and AgNPs either in sole or com- ZS (Malvern Instruments-UK). Morphology and mean bined form at different time intervals. The mechanism size of AgNPs were  determined using Field Emission of action of these agents were elucidated via estimating Scanning Electron Microscopy (FESEM) (JSM-7600F, their ability to induce oxidative stress and their effects Joel-Japan), at accelerating voltages of 15  kV as previ- on the antioxidant enzymes. Apoptosis induction abil- ously described by Alkawareek et al. [27]. ity in MCF-7 cells was examined using different staining techniques as well as cell cycle analysis. Monitoring the Antibacterial potential expression profiles of five genes namely p53, inducible Determination of minimal inhibitory concentration nitric oxide synthase (iNOS) and nuclear factor-kappaB (MIC) of the tested AgNPs against S. aureus and K. (NF-kB), B cell lymphoma-2 (Bcl-2) and microRNA-125b pnemonia was carried out using broth micro dilution (miR-125b) were also investigated following treatment. method according to the CLSI reference standards [28]. One hundred microliter of Muller-Hinton broth (MHB) (Oxoid-UK) were distributed in 96 multi-well microtiter Materials and methods plates (TPP-Swiss). Double fold serial dilutions of AgNPs Bacterial strains and culture conditions were performed. Bacterial inoculum was prepared by Clinical isolate of Staphylococcus aureus (S. aureus) and adjusting the OD at 600  nm of the previously prepared Klebsiella pneumonia (K. pneumonia) was used as a bacterial suspension to reach a turbidity of 0.5 McFarland model for Gram-positive and Gram-negative bacteria, standard which is equivalent to 10   CFU/ml. The pre - respectively. Both isolates were obtained from Kasr Al- pared suspension was further diluted and inoculated in Ainy Teaching Hospital. S. aureus was isolated from pus all plates at a final concentration of 5 × 10  CFU/ml. Posi- specimen and identified using Gram stain, biochemical tive and negative control in each plate were considered. catalase and coagulase tests in addition to formation of Plates were incubated at 37  °C for 24  h and examined golden yellow colonies on nutrient agar as well as man- visually against dark background for absence or pres- nitol fermentation on mannitol salt agar. K. pneumonia ence of turbidity. The MIC was determined as the lowest was recovered from urine specimen and the isolate was concentration of AgNPs with no visible bacterial growth cultured on blood agar and MacConkey’s agar. Colonies compared to control. Twenty µl from each well with no were identified as Klebsiella pneumonia by biochemical observed bacterial growth were further inoculated on the reaction and confirmation was carried out using the API- surface of  agar plates and incubated overnight at 37 °C. 20E (BioMérieux-France) test system according to the The lowest concentration of AgNPs that kills > 99.9% of manufacturer’s instructions. the initial bacterial inoculum is considered as the mini- The bacterial concentration of each isolate was adjusted mum bactericidal concentration (MBC). to an optical density (OD)  of 0.1 at 600  nm which is equivalent to 10 colony forming units/ml (CFU/ml) and Electromagnetic field treatment inoculated on nutrient agar plate (pH of 7.0 ± 0.2) fol- Fresh subcultures of S. aureus and K. pneumonia at a lowed by incubation for 24 h at 37 °C. Before performing final concentration of 5X10   CFU/ml were aliquoted each experiment, three colonies from each isolate  were in sterile polystyrene plastic screw capped tubes and collected from each agar plate and inoculated in 5  mL treated with different concentrations of AgNPs (2.5, 5 & nutrient broth to obtain fresh subcultures. Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 4 of 16 Hematoxylin and eosin staining 10 µM/ml) at 37 °C for 1 & 2 h interval. In the meantime, Fifty micro liters of ELF-EMF treated MCF-7 cells either another set of tubes containing the same concentrations in presence or in absence of AgNPs were dispensed on of bacteria and AgNPs were exposed to ELF-EMF of 1 m clean slides (3 slides for each treatment). Slides were Tesla for the same time interval at 37 °C. Whereas, other air-dried, methanol fixed and rehydrated using descend - tubes inoculated with the same concentration of bacteria ing concentrations of alcohol (100%, 90%, 75% and 50%). were treated with ELF-EMF in absence of AgNPs. AgNPs Slides were washed with distilled water for 5  min. The or ELF-EMF untreated bacterial suspensions were con- slides were immersed in filtered hematoxylin stain for sidered as positive  control. At the end of the treatment 3 min and washed with distilled water twice followed by period, samples were obtained from all tubes to deter- immersion in filtered eosin stain for 5 s and washed with mine the bacterial viable count. The percentage reduc - distilled water. Dried slides were immersed in xylene fol- tion in the viable count was calculated for all examined lowed by mounting with Canada balsam. The coverslips samples compared to control. were mounted to each slide and left to air dry. Micro- MCF-7 cells pre-cultured 75 cm tissue culture flasks scopic fields (100X) were photographed using digital (SPL-Korea) were dissociated using 0.25% trypsin–EDTA camera (Canon-Japan), connected to a light microscope. (Lonza-Swiss) post decanting the exhausted growth The photomicrographs were evaluated for the presence media. Detached cells were cold centrifuged (Jouan, Ki- of morphological features of apoptosis [30]. 22-France),  phosphate buffer saline (PBS) washed and resuspended in 20  ml RPMI-1640 serum free media. Apoptosis detection Cells were equally aliquoted in sterile polystyrene tubes, ELF-EMF treated MCF-7 cell suspension, mixed with treated with AgNPs I C value and exposed to ELF-EMF and without AgNPs were quantitatively examined for of 1 m Tesla for 1 and 2 h interval. Similar concentrations detection of apoptosis using annexin V-fluorescein iso - of MCF-7 cells were treated with ELF-EMF at the same thiocyanate (FITC) apoptosis detection kit (Trevigen- conditions but in absence of AgNPs. Untreated control USA). Briefly, treated and untreated cells were PBS cells were also considered. ELF-EMF treated cells either washed and collected by centrifugation. Cells were dark in presence or absence of AgNPs were examined for incubated for 15  min at room temperature with 100  μl pathological changes in addition to cell cycle and molec- annexin-V incubation reagent: 10 µl (10 X) binding buffer ular analysis as well as biochemical tests. 1, 10 µl propidium iodide (PI), 1 µl annexin V-FITC and 79 µl de-ionized water. Samples were treated with 400 μl Cytotoxicity binding buffer (1 X) and analyzed using flow cytometer Cytotoxic effect of different concentrations of AgNPs was within 1 h for maximal signal. determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyl-tetrazolium bromide (MTT) assay, where growth Cell cycle analysis media were  decanted from 96-well micro titer plates ELF-EMF treated MCF-7 cell suspension, mixed with pre-cultured with  MCF-7 cells. AgNPs were applied in and without AgNPs were processed for cell cycle analy- double fold serial dilutions to MCF-7 precultured plates. sis. Cell cycle distribution was examined by measuring Untreated wells served as negative control and plates the DNA content of nuclei labeled with propidium iodide were incubated at 37  °C for 24  h. Post incubation, the (PI). Cell suspensions were collected by cold centrifuga- plates were washed three times with PBS as 250  μl/well. tion, washed with 1 ml cold PBS, centrifuged, and fixed in Fifty μl of MTT solution (0.5 mg/ml) were added to each 70% cold ethanol (Sigma Aldrich-USA) at + 4 °C for 24 h. well and plates were incubated for further 4  h at 37  °C. Cells were re-suspended in PBS containing 40  μg/ml PI, Plates were PBS washed three times and the formed blue 0.1 mg/ml RNase and 0.1% (v/v) Triton X-100. Post dark colored formazan was dissolved using 50  μl/well DMSO incubation at 37  °C for 30  min, the cells were analyzed (Sigma Aldrich-USA) followed by shaking the plates for using flow cytometer (Becton–Dickinson, San Jose, CA, 10  min at room temperature. Optical density (OD) was USA) equipped with an argon ion laser at a wavelength measured at 570  nm using ELISA plate reader (Biotek, of 488  nm. The cell cycle and sub-G1 group were deter - ELX-800-USA). The percentage of cellular viability was mined and analyzed. calculated, and the half maximal inhibitory concentration (IC ) was determined as the concentration resulting in Real time‑PCR 50% inhibition of cellular growth following 24 h exposure Total RNA was extracted from control, 1 and 2  h ELF- to AgNPs compared to the untreated control cells using EMF treated MCF-7 cells in presence and in absence of GraphPad prism software version 5 (S. Diego-USA) [29]. AgNPs using RNeasy Mini Kit (Qiagen-USA) accord- ing to manufacturer’s instructions. The concentration M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 5 of 16 and purity of the extracted RNA was evaluated using determined via evaluation of the percentage inhibition Beckman dual spectrophotometer (Beckman-USA). rate colorimetrically. The expression level of apoptosis-related genes; P53 (F: 5’-TCA GAT CCT AGC GTC GAG CCC-3’ & R: 5’-GGG Catalase (CAT) TGT GGA ATC AAC CCA CAG-3’), BCL-2 (F: 5’-GTG Catalase enzyme activity was evaluated using an AAC TGG GGG AGG ATT GT-3’& R: 5’-GGA GAA Amplex red catalase assay kit (Molecular Probes Inc.). ATC AAA CAG AGG CC-3’), NF-kB (F: 5’-CGC ATC The principle of the assay depends on that Amplex Red CAG ACC AAC AAC A-3 & R: 5’-TGC CAG AGT TTC reagent reacts with H O in the presence of horserad- 2 2 GGT TCA C-3’), iNOS (F: 5’-AGT ATG CAA TGA ATG ish peroxidase (HRP) and catalase enzyme to produce GGG AA-3’ & R: 5’-ATT CGA TAG CTT GAG GTA highly fluorescent resorufin. The assay was carried out GA-3’), miR-125b (F: 5’-ACT GAT AAA TCC CTG AGA according to the manufacturer’s instructions. Briefly, CCC TAA C-3’ & R: 5’-TAT GGT TGT TCT GCT CTC catalase containing samples as well as control samples TGT CAC-3’)   and β-actin (F: 5’-AGA GCT ACG AGC were diluted in reaction buffer and transferred to 96-well TGC CTG AC-3’ & R: 5’-AGC ACT GTG TTG GCG microplate (25  μl/well). Hydrogen peroxide prepared as TAC AG-3’) were determined using real-time PCR. Ten 40 μM H O was added to each well (25  μl/well). The 2 2 nanograms of the extracted total RNA from each sample reaction mixture was incubated for 30 min at room tem- were used for cDNA synthesis using high capacity cDNA perature followed by adding 50 μl/well Amplex Red/HRP. reverse transcriptase kit (Thermo Fischer Scientific- The plate was incubated for 30  min at 37  °C protected USA). The obtained cDNA was subsequently amplified from light. The fluorescence intensity was evaluated in using Sybr Green I PCR master kit (Thermo Fisher Sci - all wells by reading the plate using an excitation range of entific Inc.- Lithuania) using StepOne apparatus (Applied 530–560 nm and emission at 590 nm. The activity of cat - Biosystems-Thermo Fischer Scientific), as follows: 10 min alase enzyme was determined by subtracting the sample at 95  °C for enzyme activation followed by 40 cycles value from that of negative control. of 15  s at 95  °C, 20  s at 55  °C and 30  s at 72  °C for the amplification step. Changes in the expression of the tar - Statistical analysis get genes were normalized relative to the mean critical All experiments were performed in three independ- threshold (CT) values of β-actin as a housekeeping gene. ent tests. Data were presented as the mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) and Tukey post-hoc test. Statisti- Biochemical analysis cal analysis was carried out using statistical package Assessment of reactive oxygen species, superoxide dis- for social sciences (SPSS) software (version 25), IBM, mutase and catalase levels were determined in bacterial USA. Results were considered statistically significant at models as well as in MCF-7 cancer cell model following probability < 0.05. exposure to either ELF-EMF or AgNPs in addition to the combined treatment at different time interval. Bacterial models were treated with 5 µM/ml AgNPs, while MCF-7 Results cells were treated with IC of AgNPs. Antibacterial potential Recorded data revealed that AgNPs showed greater inhibitory potentials on K. pneumonia compared to that Reactive oxygen species (ROS) in case of S. aureus, where the recorded MICs were in the Assessment of the generated reactive oxygen species was order of 3.125 and 12.5  µM/ml, respectively. It was also determined according to the manufacturer’s protocol observed that the recorded MICs exerted bactericidal using ELISA kit, Catalog No. K936-100. activity on more than 99.9% of the initial bacterial inocu- lum, so the recorded MICs were considered as MBCs. Results showed that S. aureus viable count reduction Superoxide dismutase (SOD) percentage post exposure to sole EMF for 1 and 2 h time SOD was evaluated using PromoKine kit-Catalog Num- interval was 20.4% ± 3.4 and 28.5% ± 0.9, respectively. ber: PK-CA577-K335 (PromoCell-Germany) accord- At  the same time, AgNPs exhibited a statistically sig- ing to the manufacturer’s instruction. The assay is nificant (P < 0.05) concentration dependent reduction in based on that superoxide dismutase is an antioxidative S. aureus recording 4.1% ± 0.8, 10.3% ± 1.5& 15.5% ± 3.5 enzyme which catalyzes the dismutation of the super- post 1  h exposure to 2.5, 5 & 10  µM/ml AgNPs, respec- oxide anion into hydrogen peroxide and molecular oxy- tively. Extension of the treatment period with AgNPs for gen. The rate of the reduction with a superoxide anion 2 h resulted in higher reduction in the bacterial count by is inhibited by SOD. Therefore, activity of SOD could be Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 6 of 16 recording 43.9% ± 9.7, 50% ± 5.4 and 57.3% ± 4.6, respec- 9.6% ± 1.7, 17.7% ± 2.8 and 21.8% ± 5.6, respectively. An tively. A higher reduction in the bacterial count was enhancement in the antibacterial activity was recorded observed when AgNPs were applied in combination to when AgNPs were applied in combination to EMF, EMF where they recorded 66.5% ± 12.3, 81.3% ± 1.5 and where the percentage reduction recorded 31.6% ± 2.2, 92.6% ± 7.4 post 1 h treatment, respectively. In the same 35.8% ± 4.6 & 42.3% ± 1.6 and 45.2% ± 8.3, 53.1% ± 2.5 context, maximum inhibition was recorded following 2 h and 59.4% ± 3.6 following 1 and 2  h treatment interval, exposure to 2.5 and 5  µM/ml AgNPs in combination to respectively (Fig. 1a). EMF in the order of 87.4% ± 2.3 and 97.2% ± 5.5, respec- Similar pattern of antibacterial activity was observed tively, till achieving 100% inhibition when EMF was in case of K. pneumonia but with statistically signifi - applied to the bacterial suspension in presence of 10 µM/ cant (p < 0.01) higher inhibitory potential compared to ml AgNPs (Fig. 1b). S. aureus. Treatment of K. pneumonia with EMF for 1 and 2  h resulted in reduction in  the bacterial count by 30.1% ± 2.9 and 41.3% ± 1.4, respectively. K. pneumonia Biochemical analysis in bacterial models viable count was reduced in a concentration depend- Evaluation of the ROS and SOD in S. aureus following ent manner in the order of 33.2% ± 5.3, 40.5% ± 1.8& 1 h exposure to EMF, AgNPs and EMF + AgNPs revealed 45.4% ± 5.6 post 1  h exposure to 2.5, 5 and 10  µM/ml elevated ROS levels by 0.8-, 1.0- and 1.52-fold as well as AgNPs, respectively. The inhibitory potentials were an increase in SOD by 1.12-, 0.8- and 1.98-fold compared increased by extending the incubation period to 2  h Fig. 1 1a: Evaluation of antibacterial activity of sole EMF and AgNPs as well as EMF combined with AgNPs on S. aureus viable count at 1 and 2 h time interval. 1b: Assessment of the percentage reduction of K. pneumonia viable count post 1 and 2 h exposure to EMF, AgNPs and EMF in combination to AgNPs. *P < 0.001, **P < 0.01, ***P < 0.05 M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 7 of 16 Fig. 2 a Mean fold change in ROS, SOD and CAT in S. aureus 1 and 2 h post treatment with EMF, AgNPs and EMF + AgNPs compared to control. b Biochemical analysis of ROS, SOD and CAT levels following K. pneumonia exposure to EMF, AgNPs and EMF + AgNPs at 1 and 2 h time interval. **P < 0.01, ***P < 0.05 to control, respectively. Further incubation for 2  h was accompanied by higher ROS and SOD levels in the order of 1.48-, 1.23- and 2.2-fold for ROS and 1.69-, 1.72- and 2.44-fold in case of SOD, respectively (Fig.  2a). K. pneu- monia also showed elevated levels of ROS by a value of 1.2-, 1.3- and 1.68-fold as well as 1.69-, 1.42- and 2.86- fold 1 and 2  h following exposure, respectively. In addi- tion to increased SOD levels by values of 1.17-, 1.58- and 2.0-fold as well as 1.73-, 1.58- and 2.52-fold 1 and 2  h post exposure, respectively (Fig. 2b). On the contrary, treatment of S. aureus with EMF, AgNPs and EMF + AgNPs for 1  h showed a marked Fig. 3 Evaluation of cellular viability of MCF‑7 cells post 24 h treatment with AgNPs using MTT assay indicating elevated viability reduction in CAT levels by 0.9-, 1.21- and 2.04-fold as along with decreasing AgNPs concentration. ***P < 0.05 well as 2.16-, 2.14- and 2.88-fold reduction post 2 h expo- sure (Fig.  2a). Comparable reduced levels of CAT were detected in K. pneumonia post 1  h treatment recording of apoptosis such as shrinkage of cells and peripheral 2-, 2.12- and 2.43-fold reduction as well as reduced CAT condensation of chromatin. Necrotic swollen cells with levels by 2.36-, 2.91- and 4.23-fold post 2  h treatment mixed euchromatin and heterochromatin and ruptured compared to control, respectively (Fig. 2b). cell membrane were also observed (Fig.  4e). Apoptotic shrunken cells with shrunken nuclei, peripheral con- Cytotoxicity densation of chromatin and irregular cell membranes The cytotoxic effect of AgNPs 24  h post MCF-7 cells were detected post 1 and 2  h treatment with AgNPs, treatment was determined using MTT assay. Recorded respectively (Fig.  4b, f ). Treatment with ELF-EMF in data revealed that viability was concentration dependent, combination to AgNPs for 1  h revealed the presence where the  viability increases as long as the concentra- of swollen necrotic cells and swollen nuclei with mixed tions of AgNPs decrease till reaching 100% viability at a euchromatin and heterochromatin and ruptured cell concentration of 1  μM/ml. The calculated IC value of membranes. Shrunken apoptotic cells with peripheral AgNPs was 4.15 ± 0.20 μM/ml (Fig. 3). condensation of chromatin in addition to secondary necrotic cells with peripheral condensation of chroma- Hematoxylin and eosin staining tin and ruptured cell membranes as well as apoptotic Microscopic examination of MCF-7 cells treated with bodies were also observed (Fig.  4c). Necrotic cells with sole ELF-EMF for 1  h revealed the detection of swol- mixed euchromatin and heterochromatin, ruptured cell len cells, swollen nuclei with mixed euchromatin and membranes, intranuclear eosinophilic structures as well heterochromatin, ruptured cell membranes as well as as shrunken apoptotic cells with irregular cellular and intranuclear eosinophilic structures (Fig. 4a). Extending nuclear membranes were detected post 2 h exposure to the exposure time to 2 h showed characteristic features ELF-EMF in combination to AgNPs (Fig.  4g). On the Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 8 of 16 Fig. 4 Pathological changes detected post MCF‑7 cells exposure to sole ELF‑EMF, AgNPs and ELF‑EMF in combination to AgNPs at different time interval using hematoxylin and eosin staining. a MCF‑7 cells photomicrograph post 1 h exposure to ELF‑EMF showing swollen cells, swollen nuclei with mixed euchromatin and heterochromatin (Yellow arrows) as well as ruptured cell membranes (Green arrows) in addition to intranuclear eosinophilic structures (Red arrows). e Photomicrographs following 2 h treatment with ELF‑EMF revealing the occurrence of apoptosis as indicated by the observed shrunken apoptotic cells with peripheral condensation of chromatin (Green arrows) as well as necrotic cells with mixed euchromatin and heterochromatin (Yellow arrows) and swollen cell with ruptured cell membrane (Red arrow). b&f Photomicrographs of MCF‑7 cells post exposure to AgNPs for 1 and 2 h, respectively revealing the presence of shrunken apoptotic cells with shrunken nuclei (Yellow arrows), peripheral condensation of chromatin (Green arrows) and irregular cell membranes (Red arrows). c Swollen necrotic MCF‑7 cells and swollen nuclei with mixed euchromatin and heterochromatin and ruptured cell membranes (Blue arrows) were detected post 1 h exposure to ELF‑EMF in combination to AgNPs. Shrunken apoptotic cell (Green arrow) with peripheral condensation of chromatin (Orange arrow) in addition to secondary necrotic cells with peripheral condensation of chromatin and ruptured cell membranes (yellow arrow) as well as apoptotic bodies (Red arrows) were also observed. g Photomicrograph post 2 h exposure to ELF‑EMF in combination to AgNPs showing necrotic cells with mixed euchromatin and heterochromatin (Red arrows), ruptured cell membranes (Green arrows), intranuclear eosinophilic structures (Yellow arrows) and shrunken apoptotic cells with irregular cell and nuclear membranes (Black arrows). d&h Control untreated MCF‑7 cells examined at 1 and 2 h, respectively showing regular cells with hyperchromatic nuclei (Original magnification 100X, Oil) contrary, untreated cells showed regular appearance 8.26% ± 1.0) and AgNPs (10.45% ± 4.2 and 7.65% ± 0.7), with hyperchromatic nuclei, respectively (Fig. 4d, h). respectively (Fig. 5). Apoptosis Cell cycle analysis Recorded data showed that the sole exposure to Recorded data revealed a time dependent apoptosis ELF-EMF and AgNPs or in combination signifi - as indicated by the elevation of MCF-7 cells in Pre G1 cantly (P < 0.001) induced time dependent apopto- phase (16.85% ± 2, 24.11% ± 3.1) post treatment with sis compared to untreated cells. Also, a statistically ELF-EMF for 1 and 2  h, respectively. Treatment with significant (P < 0.01) induction of apoptosis post 1  h AgNPs resulted in a similar pattern of apoptosis induc- exposure to ELF-EMF-AgNPs combined form record- tion recording 14.34% ± 5.5, 20.35% ± 0.8 of cells in Pre ing 16.65% ± 1.3 and 11.48% ± 1.3 of the analyzed cells G1 phase post 1 and 2  h exposure, respectively. In the in late apoptosis and necrosis, respectively, compared same context, MCF-7 cell treatment with ELF-EMF in to lower levels of the detected cells (5.61% ± 1.8 and combination to AgNPs induced a statistically significant 5.87% ± 2.1) post treatment with sole ELF-EMF or (p < 0.001) time dependent higher levels of apoptotic AgNPs (7.82% ± 1.1 and 6.29% ± 3.8), respectively. Fur- cells (22.45% ± 3.5, 38.26% ± 5.4) post 1 and 2  h treat- ther exposure to ELF-EMF combined with AgNPs for ment, respectively. Also, sole treatment with ELF-EMF 2  h significantly (P < 0.01) resulted in a marked eleva- induced a significant (p < 0.01) time dependent G2/M tion of early apoptotic and necrotic cells in the order phase arrest (24.52% ± 2.2, 35.53% ± 1.8) compared to of 21.84% ± 2.5 and 13.71% ± 1.8 compared to that in untreated cells (18.95% ± 1.5, 20.76% ± 3.2). Whereas case of treatment with sole ELF-EMF (11.59% ± 1.5 and significant (p < 0.01) arrest during S phase (40.68% ± 7.1, 51.24% ± 2.2) was observed 1 and 2  h post exposure M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 9 of 16 Fig. 5 Evaluation of early and late apoptosis as well as necrosis post MCF‑7 cells post exposure to ELF‑EMF, AgNPs and ELF‑EMF/AgNPs for 1 and 2 h interval using annexin V‑FITC apoptosis detection kit. *P < 0.001, **P < 0.01, ***P < 0.05. a necrotic cells, b late apoptosis, c early apoptosis to sole AgNPs, respectively. Similarly, combined treat- observed post 1 h exposure to combined treatment com- ment with ELF-EMF and AgNPs resulted in a significant pared to that recorded 2 h post treatment recording 0.42- (p < 0.05) S phase arrest (49.43% ± 4.6, 55.17% ± 3.1) 1 and 0.55-fold change, respectively (Fig. 7). and 2 h post treatment, respectively (Fig. 6). Biochemical analysis in MCF‑7 cells The oxidative stress induced 1 h post exposure of MCF-7 Gene expression profile cells to ELF-EMF, AgNPs and ELF-EMF combined with Expression level of apoptosis related genes was evaluated, AgNPs showed statistically significant (P < 0.05) elevated it was found that p53, iNOS and NF-kB genes showed levels of the generated ROS recording 1.18- 1.0- and 2.04- significant up-regulation recording 4.86-, 3.47- and 5.98- fold compared to untreated cell control, respectively. fold increase post 1  h exposure compared to 6.6-, 4.43- Whereas, extending the exposure time to 2 h resulted in and 7.37-fold elevation post 2  h treatment, respectively. increased levels of ROS in the order of 1.45-, 1.72- and Down-regulation in the expression level of Bcl-2 and 2.88-fold compared to untreated cell control, respectively. miRNA-125b in the order of 0.69- and 0.68-fold was Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 10 of 16 Fig. 6 Evaluation of cell cycle profile post short ‑term exposure of MCF‑7 cells to ELF‑EMF and AgNPs as sole treatment as well as in combination at 1 and 2 h time interval using flow cytometry. *P < 0.001, **P < 0.01, ***P < 0.05 Fig. 7 Evaluation of the expression profile of apoptosis related genes in MCF‑7 cells post sole exposure to ELF‑EMF and AgNPs as well as in combination at different time interval using real time PCR. **P < 0.01, ***P < 0.05 Recorded data showed a significant (P < 0.05) elevated Discussion SOD levels recording 1.55-, 1.43- and 2.33-fold increase Multi-drug resistance is a threatening obstacle in the 1 h post treatment with ELF-EMF, AgNPs and ELF-EMF/ treatment of infectious diseases, where the misuse of AgNPs, respectively. While MCF-7 cells following 2  h broad-spectrum antibiotics has evoked antibiotic resist- treatment exhibited higher enhancement of SOD activ- ance among several human bacterial pathogens. AgNPs ity in the order of 2.06-, 1.8- and 2.43-fold compared to as well as ELF-EMF have attracted much attention in this untreated cells, respectively. On the other side, the activ- field due to their recorded antibacterial potentials [6]. ity of catalase was significantly (P < 0.05) reduced post 1 h u Th s, the present study tried to highlight the antibacte - treatment with ELF-EMF, AgNPs and ELF-EMF/AgNPs rial potentials developed following exposure to these two by 2.04-, 1.17- and 2.66-fold, respectively. In the same weapons either alone or in combination. For this issue, context, the activity of catalase was also reduced post 2 h S. aureus and K. pneumonia were selected as models for treatment by 2.23-, 2.0- and 4.0- fold, respectively (Fig. 8). Gram-positive and Gram-negative bacteria, respectively. S. aureus is a commensal bacterium known to asymp- tomatically colonize the human skin, nasal passages, and gastrointestinal tract. S. aureus infections range from mild skin and soft tissue infections to more severe M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 11 of 16 Fig. 8 Assessment of the generation of ROS, SOD and CAT post MCF‑7 cells exposure to ELF‑EMF and AgNPs as sole treatment as well as in combination at 1 and 2 h time interval. **P < 0.01, ***P < 0.05 invasive diseases, such as endocarditis, bacteremia, sep- cytoplasm as well as degrading the chromosomal DNA sis, pneumonia, and osteomyelitis [31]. In the meantime, which subsequently result in failure of ATP produc- K. pneumonia accounts for a significant proportion of tion and chromosomal replication [33]. Another study hospital-acquired urinary tract infections, pneumonia, examined E. coli post exposure to AgNPs using trans- septicemia as well as soft tissue infections and it has mission electron microscope to observe the interaction become multi-resistant to various types of antibiotics. between them. It was found that the positively charged k. pneumonia infections are also of clinical importance Ag ions were attracted to the negatively charged bac- among patients in intensive care units with compromised terial lipopolysaccharides, induce the formation of immune systems such as cancer patients [6]. The present holes in the bacterial cell wall and result in cell lysis study aimed also to investigate the influence of short- [34]. It is important to note that the rate of dissolution term exposure to ELF-EMF and AgNPs either in sole of AgNPs to release Ag + ions is multifactorial, where it or combined form at different time intervals. Thus, we is dependent on several factors including their physico- selected 1 and 2 h time interval as a model for the short- chemical properties such as the size, shape, concentra- term exposure time. tion, capping agent and colloidal state of NPs as well as Similar to the current findings it was reported that the presence of chlorine, thiols, sulfur, and oxygen [35]. AgNPs exhibited antibacterial activity against E. coli In the current study, the recorded MICs and the in a concentration and time dependent manner at a viable count following treatment with AgNPs revealed range of low concentrations in the order of 10  µM and that these particles showed greater inhibitory poten- 100 µM [32]. Many other studies demonstrated that the tials towards Gram-negative bacteria compared to that antibacterial potential of AgNPs isn’t only specific for observed in case of Gram-positive bacteria. Similar Gram-negative bacteria (E. coli, K. pneumonia and P. findings were reported in another study, where they aeruginosa), however its activity also extents to Gram demonstrated higher antibacterial activity of AgNPs positive (S. aureus and B. subtilis) bacterial strains. It against E. coli compared to that exerted on S. aureus was reported that the cytotoxic effect of AgNPs against [36]. That was attributed to the structural differences in bacteria may result from the oxidative dissolution of the composition of the cell walls between Gram-posi- AgNPs and the release of Ag ions from AgNPs. The tive and Gram-negative bacteria. Gram-negative bacte- released Ag ions interact with sulfhydryl (-SH) groups ria have an outer layer of lipopolysaccharides and thin of cell wall-bound enzymes and proteins, then inter- layer of peptidoglycan which confers for the overall lack fere with the respiratory chain of bacteria resulting in of rigidity of the cell wall of Gram-negative bacteria. disruption of the bacterial cell wall. In addition to the On the other side, Gram-positive bacteria have a three- ability of the released ions to penetrate the bacterial dimensional rigid cell wall consisting of thicker layer of cell wall and react with thiol groups of the proteins in peptidoglycan which is formed of linear polysaccharide Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 12 of 16 chains cross-linked by short peptides. The rigidity and due to the production of ROS. It was reported that the cross-linking not only diminish the cell wall attachment positively charged silver ions released from AgNPs could sites for AgNPs in Gram-positive bacteria but also induce oxidative stress in bacterial cells due to their increase the difficulty of AgNPs penetration to the bac - interference with the normal function of the bacterial terial cell wall [19]. electron transport chain and thus facilitating the genera- In the same context to the antibacterial potentials of tion of ROS. ROS generation is primarily responsible for AgNPs, other studies demonstrated the bacterial inhibi- the bacterial death as it enhances lipid peroxidation but tory activity of ELF-EMF. It was found that exposure of hindered ATP production and DNA replication [40]. At different Gram-positive and Gram-negative bacterial the same time, another study attributed the antibacte- strains to ELF-EMF resulted in reduction in their growth rial potential of AgNPs to the combined effect of these rates with respect to control samples. In addition to the particles on the bacterial components as previously dem- ultrastructural changes that were observed post treat- onstrated in addition to their ability to induce oxidative ment with this type of electromagnetic waves [37]. Also, stress [41]. another study reported a reduction in the percentage It is important to point out that, SOD and CAT are viability of S. aureus and E. coli post exposure to ELF- antioxidant enzymes which have been identified as criti - EMF. That was justified by the ability of these waves to cal modulators in AgNPs induced oxidative stress and alter the structure and function of the ion channels and are considered as bioindicators of increased ROS pro- efflux pumps of bacterial cell walls resulting in an altera - duction. [42]. AgNPs induced oxidative stress in bacte- tion in the permeability of the bacterial cell wall to dif- rial cells results in the generation of superoxide anion •− ferent molecules leading to cell death. Although many (O ). This free radical could be dismuted to hydrogen researchers tried to explore the effect of EMF on bacte -peroxide (H O ) by the effect of SOD enzyme. The pro - 2 2 ria, but these effects are variable depending on the fre -duced H O is then quickly converted to H O and O by 2 2 2 2 quency and intensity of EMF, exposure time as well as CAT enzyme. The H O produced by the effect of SOD 2 2 the phase of bacterial growth, ingredients of the media, could penetrate the bacterial membranes and interacts 2+ genetic properties, presence or absence of oxygen, and with ferrous ion (F e ) and thiol groups (-SH) of protein bacterial membrane features [38]. In accordance with the cysteines leading to inactivation of essential enzymes of 2+ current findings, it was demonstrated that the antibacte - the pathogen. Moreover, F e is oxidized during a Fenton rial potentials of either AgNPs or EMF were highly effec - reaction by H O and generates hydroxyl radical, which 2 2 tive against Gram-negative bacteria compared to that in turn causes further destruction in the bacterial pro- recorded in case of Gram-positive [2]. teins, DNA, and lipids [43]. In agreement with the present study, it was reported Similar to our results, a study found that the activity of that EMF is a versatile tool which could be successfully SOD in AgNPs treated P. aeruginosa was elevated, how- used for increasing the susceptibility of bacteria to anti- ever the activity of CAT was reduced post treatment. bacterial agents. They reported elevated antibacterial This study reported that AgNPs enhanced the activity of potentials when iron NPs were applied in combination to SOD enzyme resulting in an accumulation of H O . On 2 2 EMF against Bacillus subtilis and E. coli bacterial mod- the other hand, AgNPs treated cells failed to get rid of els. They attributed this elevated inactivation efficiency to the accumulated H O due to suppression of CAT activ- 2 2 the induction of higher local field gradients, hyperther - ity by the effect of AgNPs. Accordingly, AgNPs treated mia, and motion of both the bacterial cells and magnetic bacterial cells showed elevated levels of cell death due to NPs. Despite the ability of EMF to enhance the antibac- the enhancement of oxidative stress by the effect of the terial potentials of different antimicrobials such as NPs, accumulated H O [34]. That was suggested to be related 2 2 but their exact mechanism of action is still unclear and to the formation of AgNPs-CAT complex which in turn requires further investigations [39]. Thus, the present resulted in conformational changes in CAT enzyme study evaluated the levels of ROS as well as the antioxi- leading to an impairment of its enzymatic activity. In dant enzymes (SOD and CAT) to explore the reason for contrast, the formation of AgNPs-SOD complex has no the bacterial inhibitory potentials of AgNPs and ELF- influence on its enzymatic activity [44]. EMF either alone or in combination. Current findings Regarding the anticancer potential of AgNPs and in revealed that the antibacterial potential of AgNPs against agreement with the present findings, it was reported S. aureus and K. pneumonia was directly proportional that AgNPs reduced MCF-7 cellular viability in a to the detected elevated levels of ROS in both bacterial dose  dependent manner recording an IC value of models post treatment. Consequently, the recorded bac- 6.28 μM [45]. It was reported that excessive production terial inhibitory potentials could be justified by the abil - of ROS in cells by direct interaction with AgNPs and/ ity of AgNPs to induce oxidative stress in bacterial cells or dissolved silver ions is currently accepted as one of M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 13 of 16 the main mechanisms of cellular toxicity of engineered PI staining. Also, a significant time dependent elevated nanoparticles in living organisms. Although ROS have late apoptosis was detected  following cellular treatment many signaling and information functions, but it could with EMF-AgNPs combination compared to  that  in also diminish the antioxidant defense system leading case of sole application of EMF and AgNPs as well as to to damage of DNA, lipids and proteins [44]. That was untreated cell control. obvious in the current study, where the recorded results Cell cycle analysis in addition to  the assessment of the revealed a time dependent reduction in CAT enzyme gene expression profile of 5 apoptosis related genes were activity, which is considered an antioxidant enzyme, also performed to explore the correlation between the post treatment with AgNPs either alone or in combi- detected apoptosis and the influence of the applied treat - nation to EMF. The observed CAT enzymatic activity ments on cell cycle phases as well as the gene expression reduction was also in parallel to apoptosis induction pattern. Recorded results proved the potential of EMF, following treatment with AgNPs as well as production AgNPs and  their combination to induce apoptosis as of ROS. This might be attributed to AgNPs induced they significantly induced elevated level of cells in Pre G1 generation of oxidative stress which was intensified by phase. The exposure to AgNPs was also accompanied by reduction of CAT enzymatic activity resulting in H O S phase arrest. On the other hand, following the exposure 2 2 accumulation as previously explained. to EMF there was a significant elevation in the percent - The recorded ability of EMF to induce apoptosis in age of cells in G2/M phase. In agreement with the current MCF-7 cells could be related to some reports that  high- findings, it was reported that treatment of MCF-7 cells lighted the ability of electromagnetic field to induce with an I C concentration of AgNPs generated progres- hyperthermia in tumor cells. It was demonstrated that sive accumulation of cells in the S phase of the cell cycle hyperthermia could kill cancer cells but with limited haz- [45]. Another study reported the accumulation of MCF-7 ards on healthy cells due to the well-known biophysical cells in the G2/M phase 6, 12 and 24  h following expo- differences between the cancer cells and their healthy sure to EMF [49]. counterparts. Hyperthermia not only induce apoptosis Regarding investigating the mechanism of action of in cancer cells but may also enhance the susceptibility of the EMF and AgNPs on apoptosis at the molecular level. cancer cells to anticancer agents thus allowing the reduc- The expression levels of two pro-apoptotic genes (p53 tion of their administered doses [46]. That was obvious in and iNOS) as well as two anti-apoptotic genes (Bcl-2 and the present findings, where the current results reported miRNA-125b) in addition to the impact on the expres- a time dependent ability of the combination between sion level of NF-kB were evaluated following exposure EMF and AgNPs to enhance apoptosis in AgNPs treated to different treatments. In agreement with the current MCF-7 cells compared to the lower apoptosis induced in findings, a recent study reported that the expression lev - case of sole application of AgNPs. els of pro-apoptotic genes (p53, Bax and caspase-3) were In accordance with the current findings, a study inves - significantly up-regulated, whereas the expression of the tigated the biochemical consequences following exposure anti-apoptotic gene Bcl-2 was significantly down-regu - to EMF and suggested that the genotoxic events associ- lated in AgNPs treated MCF-7 and colon cancer (HCT- ated with exposure to EMF might be due to its ability 116) cells [50]. to elevate the levels of free radicals which in turn led to The current study aimed to explore other novel mecha - DNA damage [47]. Another in  vivo study demonstrated nisms that might be involved in AgNPs or EMF induced alteration in different oxidative stress biomarkers (SOD cytotoxicity on MCF-7 cells. Thus, the expression profile and CAT) as well as increased ROS levels in the brain of of iNOS gene was investigated. Similar reports revealed male rats following exposure to ELF-EMF for 2 h. It was that AgNPs elevated the expression level of iNOS genes also found that the exposure of murine squamous cell as well as nitric oxide (NO) derived reactive species in carcinoma line (AT478) to ELF-EMF for 16 min resulted human osteoblast cells [51]. However, it is essential to in an increased ROS formation and SOD activities. Simi- prove the generation of NO in our study on the transi- lar observations showed elevated ROS formation and tional level not only on the molecular level. In accordance induced cell death post 1  h exposure of breast cancer with the current findings, a study demonstrated down- (MDA-MB-231) cells to EMF [48]. regulation in the expression levels of various miRNA The ability of AgNPs and EMF to induce apoptosis genes following treatment of cancer liver (HepG2) cells either alone or in combination was proved in the pre- with different types of nanoparticles. The reported sent study using several techniques. For example, hema- changes were highly induced by AgNPs followed by gold toxylin and eosin staining indicated the enhancement of and iron nanoparticles and at  the same time they were apoptosis in MCF-7 cells. These results were confirmed accompanied by inhibition of both cellular proliferation via flowcytometric analysis using annexin V-FITC and and tumorigenesis [52]. Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 14 of 16 It is worth to point out that NF-kB gene plays a dual exposure time. The recorded reduction in the bacterial role in apoptosis, where it could function as both a pro- viable count following exposure to these agents was apoptotic and anti-apoptotic regulatory factor within higher against Gram-negative bacteria as compared a single cell type [53]. It was also reported that the pro- to Gram-positive bacterial model. These antibacterial apoptotic or anti-apoptotic function of NF-kB is deter- potentials were suggested to be related to the capa- mined by the nature of the apoptotic stimulus. u Th s, bility of these agents to induce oxidative stress by the the nature of the signals evoked by the respective death generation of ROS. However, their effect was magnified enhancers determines whether NF-kB induction leads via enhancing the antioxidant activity of SOD and on to apoptosis or survival, suggesting that the modulation the other side reducing the activity of catalase enzyme of NF-kB activity may present a new approach in can- resulting in elevated toxicity that might be attributed cer adjuvanted therapy [54]. A study demonstrated that to H O accumulation. Consequently, the combination 2 2 the NF-kB pathway has been proposed to be a key fac- between the tested agents could present a novel strat- tor contributing to the unusual phenotype and aggres- egy for infection control and to overcome bacterial siveness of breast cancer. In agreement with our results, resistance. In the meantime, a time dependent induc- it was reported that some up-regulated NF-kB-related tion of apoptosis was observed following treatment of genes could serve as novel therapeutic targets in breast MCF-7 cells with AgNPs, ELF-EMF as well as in com- cancer [55]. bination. That was proposed to the ability of the tested To the best of our knowledge, this is the first study that treatments to significantly elevated the Pre G1 apop - tried to explore the apoptosis induction potentials of the totic phase of MCF-7 cells. Moreover, the exposure to combination between ELF-EMF and AgNPs. Also, the AgNPs induced S phase arrest, whereas the EMF treat- mechanisms that may be involved in the antibacterial and ment was accompanied by accumulation of cells in the anticancer potentials of the short-term exposure to ELF- G2/M phase. Additionally, up-regulation in the expres- EMF in combination to AgNPs have not been previously sion level of p53, iNOS and NF-kB genes was observed, investigated. Current findings strongly suggest that the however down-regulation of the anti-apoptotic genes, ability of ELF-EMF in combination to AgNPs to induce namely Bcl-2 and miRNA-125b was detected post oxidative stress in bacterial and cancer cells via genera- treatment. Biochemical analysis also shed light on the tion of ROS, SOD induction and catalase reduction could ability of both EMF and AgNPs to induce apoptosis via be responsible for their antibacterial and anticancer generation of oxidative stress. Finally, it could be con- potentials. The significance of this study isn’t only related cluded that AgNPs and ELF-EMF either in sole applica- to exploring the antibacterial and the anticancer poten- tion or in combination could be considered as potential tials of AgNPs and ELF-EMF either alone or in combi- oxidative stress generating agents that might pave the nation as new therapeutic approaches, but it spotlight way to solve the problem of antibiotic resistance espe- on the effectiveness of the combination between these cially in immunocompromised cancer patients and agents as an essential life-saving approach if this type of could successfully direct cancer cells to death. treatments could be applied clinically. That might offer greater health improvement especially in the immuno- Abbreviations compromised cancer patients who are more vulnerable AgNPs: Silver nanoparticles; Bcl‑2: B cell lymphoma‑2; CAT : Catalase; ELF‑EMF: to develop infections with antibiotic resistant pathogens. Extremely low frequency electromagnetic field; FITC: Fluorescein isothiocy‑ anate; iNOS: Inducible nitric oxide synthase; miR‑125b: MicroRNA‑125b; NF‑kB: Nuclear factor‑kappaB; PI: Propidium iodide; ROS: Reactive oxygen species; Study limitations SOD: Superoxide dismutase. This research study was performed on  only two kinds Acknowledgements of pathogenic bacterial models, one cancer cell model Not applicable and only at one tested intensity (1  m Tesla) of the EMF. Authors’ contributions Further studies on different bacterial and cancer cell AFM: conceptualization, methodology, resources, investigation, software, writ‑ models as well as different exposure conditions are ing‑ original draft. MN: resources, methodology, investigation, writing review. recommended. MEA: resources, methodology, investigation, writing review. TMMA: resources, methodology, investigation, writing review. WMA‑E: resources, methodology, investigation, writing review. HFK: resources, methodology, investigation, Conclusions writing review. EERM: resources, methodology, investigation, writing review. LEEM: resources, methodology, investigation, writing review. TAG: resources, AgNPs and ELF-EMF could be considered as poten- methodology, investigation, writing review. MAE‑FD: resources, methodology, tial antibacterial and anticancer agents. The activities investigation, writing review. RIS: conceptualization, methodology, resources, of these agents were enhanced upon their combina- investigation, software, writing‑ original draft, writing‑review, editing and submitting the paper to the journal. All authors read and approved the final tions in a time dependent manner even though at short manuscript. M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 15 of 16 Funding 12. Tofani S, Barone D, Berardelli M, Berno E, Cintorino M, Foglia L, Ossola P, This research received no specific grant from any funding agency in the pub ‑ Ronchetto F, Toso E, Eandi M. Static and ELF magnetic fields enhance the lic, commercial, or not‑for ‑profit sectors. in vivo anti‑tumor efficacy of cis‑platin against lewis lung carcinoma, but not of cyclophosphamide against B16 melanotic melanoma. Pharmacol Availability of data and materials Res. 2003;48(1):83–90. All data generated or analyzed during this study are included in this published 13. Akbarnejad Z, Eskandary H, Dini L, Vergallo C, Nematollahi‑Mahani SN, article. Farsinejad A, Abadi MF, Ahmadi M. Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low‑frequency electromagnetic field (100 Hz, 100 G). Declarations Biomed Pharmacother. 2017;1(92):254–64. 14. Sriram MI, Kanth SB, Kalishwaralal K, Gurunathan S. Antitumor activity Ethical approval and consent to participate of silver nanoparticles in Dalton’s lymphoma ascites tumor model. Int J Not applicable as the study didn’t include human participants. Nanomedicine. 2010;5(5):753–62. https:// doi. org/ 10. 2147/ IJN. S11727. PMID: 21042 421; PMCID: PMC29 62271. Consent for publication 15. Banerjee I, Pangule RC, Kane RS. Antifouling coatings: recent develop‑ Not applicable. ments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms. Adv Mater. 2011;23(6):690–718. Competing interests 16. Sanpui P, Murugadoss A, Prasad PD, Ghosh SS, Chattopadhyay A. The The authors declare that they have no competing interests. antibacterial properties of a novel chitosan–Ag‑nanoparticle composite. Int J Food Microbiol. 2008;124(2):142–6. Author details 17. Gomathi AC, Rajarathinam SX, Sadiq AM, Rajeshkumar S. Anticancer activ‑ International Center for Training and Advanced Researches (ICTAR‑Egypt), ity of silver nanoparticles synthesized using aqueous fruit shell extract of Cairo, Egypt. Histology Department, Faculty of Medicine, Al‑Azhar University, Tamarindus indica on MCF‑7 human breast cancer cell line. J Drug Deliv Cairo, Egypt. Histology Department, Faculty of Medicine, Al‑Azhar Univer ‑ Sci Technol. 2020;55:101376. sity, Damietta, Egypt. Pathology Department, Faculty of Medicine, Al‑Azhar 18. Gurunathan S, Lee KJ, Kalishwaralal K, Sheikpranbabu S, Vaidyanathan R, University, Cairo, Egypt. Microbiology and Immunology Department, Faculty Eom SH. Antiangiogenic properties of silver nanoparticles. Biomaterials. of Pharmacy, Ahram Canadian University (ACU), 4th Industrial Zone, Banks 2009;30(31):6341–50. Complex, 6th October City, Cairo, Egypt. 19. Franci G, Falanga A, Galdiero S, Palomba L, Rai M, Morelli G, Galdiero M. Silver nanoparticles as potential antibacterial agents. Molecules. Received: 26 October 2021 Accepted: 14 January 2022 2015;20(5):8856–74. 20. AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxic‑ ity and genotoxicity of silver nanoparticles in human cells. ACS Nano. 2009;3(2):279–90. 21. Franco‑Molina MA, Mendoza‑ Gamboa E, Sierra‑Rivera CA, Gómez‑Flores References RA, Zapata‑Benavides P, Castillo ‑ Tello P, Alcocer‑ González JM, Miranda‑ 1. Malik B, Bhattacharyya S. Antibiotic drug‑resistance as a complex system Hernández DF, Tamez‑ Guerra RS, Rodríguez‑Padilla C. Antitumor activity driven by socio‑ economic growth and antibiotic misuse. Sci Rep. of colloidal silver on MCF‑7 human breast cancer cells. J Exp Clin Cancer 2019;9(1):9788. Res. 2010;29(1):1–7. 2. El‑Kaliuoby MI, Khalil AM, El‑Khatib AM, Shehata N. Antibacterial syner ‑ 22. Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ. The apoptotic effect gism of electrospun nanofiber mats functioned with silver nanoparticles of nanosilver is mediated by a ROS‑and JNK ‑ dependent mechanism and pulsed electromagnetic waves. Polymers. 2021;13(2):277. involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett. 3. Chan K, Morris GJ. Chemoprevention of breast cancer for women at high 2008;179(3):130–9. risk. Semin Oncol. 2006;33(6):642–6. https:// doi. org/ 10. 1053/j. semin oncol. 23. Raja G, Cao S, Kim DH, Kim TJ. Mechanoregulation of titanium dioxide 2006. 08. 017 (PMID: 17145342). nanoparticles in cancer therapy. Mater Sci Eng C. 2020;107:110303. 4. Johnston SR. Acquired tamoxifen resistance in human breast can‑ 24. Raja G, Selvaraj V, Suk M, Suk KT, Kim TJ. Metabolic phenotyping analysis cer–potential mechanisms and clinical implications. Anticancer Drugs. of graphene oxide nanosheets exposures in breast cancer cells: metabo‑ 1997;8(10):911–30. lomics profiling techniques. Process Biochem. 2021;1(104):39–45. 5. Brown K. Breast cancer chemoprevention: risk‑benefit effects of the 25. Raja G, Jung Y, Jung SH, Kim TJ. 1H‑NMR‑based metabolomics for cancer antioestrogen tamoxifen. Expert Opin Drug Saf. 2002;1(3):253–67. targeting and metabolic engineering–a review. Process Biochem. 2020. 6. El‑kaliuoby MI, Khalil AM, El‑Khatib AM, Shalaby TI. Synergistic antibacte ‑ 26. Raja G, Jang YK, Suh JS, Kim HS, Ahn SH, Kim TJ. Microcellular environ‑ rial effect of silver nanoparticles and extremely low‑frequency pulsed mental regulation of silver nanoparticles in cancer therapy: a critical magnetic fields on klebsiella pneumoniae. Communications. 2018;15:23. review. Cancers. 2020;12(3):664. 7. Segatore B, Setacci D, Bennato F, Cardigno R, Amicosante G, Iorio R. 27. Alkawareek MY, Bahlool A, Abulateefeh SR, Alkilany AM. Synergistic Evaluations of the effects of extremely low‑frequency electromagnetic antibacterial activity of silver nanoparticles and hydrogen peroxide. PLoS fields on growth and antibiotic susceptibility of escherichia coli and ONE. 2019;14(8):e0220575. pseudomonas aeruginosa. Int J Microbiol. 2012;2012:587293. https:// doi. 28. Wayne PA, Clinical and laboratory standards institute. Performance stand‑ org/ 10. 1155/ 2012/ 587293. ards for antimicrobial susceptibility testing. twenty‑first informational 8. Falone S, Santini S Jr, Cordone V, Di Emidio G, Tatone C, Cacchio M, Ami‑ supplement. M100–S21. CLSI. 2011: 100–121. carelli F. Extremely low‑frequency magnetic fields and redox ‑responsive 29. Vijayakumar S, Ganesan S. In vitro cytotoxicity assay on gold nanoparti‑ pathways linked to cancer drug resistance: insights from co‑ exposure‑ cles with different stabilizing agents. J Nanomater. 2012. https:// doi. org/ based in vitro studies. Front Public Health. 2018;23(6):33. 10. 1155/ 2012/ 734398 9. Tofani S. Electromagnetic field exposure system for the study of possible 30. Sholqamy MI, Abd‑ElHamid ES, Mostafa AH, Mohamed AF, El‑Said WA. anti‑ cancer activity. IEEE Trans Electromagn Compat. 2002;44(1):148–51. Monitoring the anticancer effects of two different gold nanostructures 10. Oh IR, Raymundo B, Jung SA, Kim HJ, Park JK, Kim CW. Extremely low‑ shapes towards Hep‑2 Cells. Int J Med Nano Res. 2019;6:028. https:// doi. frequency electromagnetic field altered PPARγ and CCL2 levels and sup ‑ org/ 10. 23937/ 2378‑ 3664. 14100 28. pressed CD44+/CD24− breast cancer cells characteristics. Bull Korean 31. Jenkins A, Diep BA, Mai TT, Vo NH, Warrener P, Suzich J, Stover CK, Chem Soc. 2020;41(8):812–23. Sellman BR. Differential expression and roles of Staphylococcus 11. Ashdown CP, Johns SC, Aminov E, Unanian M, Connacher W, Friend J, Fus‑ aureus virulence determinants during colonization and disease. MBio. ter MM. Pulsed low‑frequency magnetic fields induce tumor membrane 2015;6(1):e02272‑ e2314. disruption and altered cell viability. Biophys J. 2020;118(7):1552–63. 32. Orlov IA, Sankova TP, Babich PS, Sosnin IM, Ilyechova EY, Kirilenko DA, Brunkov PN, Ataev GL, Romanov AE, Puchkova LV. New silver Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 16 of 16 nanoparticles induce apoptosis‑like process in E. coli and interfere with antiapoptotic regulatory factor within a single cell type. Cell Death Differ. mammalian copper metabolism. Int J Nanomed. 2016;11:6561. 1999;6(6):570–82. 33. Liao C, Li Y, Tjong SC. Bactericidal and cytotoxic properties of silver nano‑ 54. Kaltschmidt B, Kaltschmidt C, Hofmann TG, Hehner SP, Dröge W, Schmitz particles. Int J Mol Sci. 2019;20(2):449. ML. The pro‑ or anti‑apoptotic function of NF‑κB is determined by the 34. Liao S, Zhang Y, Pan X, Zhu F, Jiang C, Liu Q, Cheng Z, Dai G, Wu G, Wang nature of the apoptotic stimulus. Eur J Biochem. 2000;267(12):3828–35. L, Chen L. Antibacterial activity and mechanism of silver nanoparticles 55. Lerebours F, Vacher S, Andrieu C, Espie M, Marty M, Lidereau R, Bieche I. against multidrug‑resistant Pseudomonas aeruginosa. Int J Nanomed. NF‑kappa B genes have a major role in inflammatory breast cancer. BMC 2019;14:1469. Cancer. 2008;8(1):1–1. 35. Ferdous Z, Nemmar A. Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure. Int J Publisher’s Note Mol Sci. 2020;21(7):2375. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 36. Schiffman JD, Wang Y, Giannelis EP, Elimelech M. Biocidal activity lished maps and institutional affiliations. of plasma modified electrospun polysulfone mats functionalized with polyethyleneimine‑ capped silver nanoparticles. Langmuir. 2011;27(21):13159–64. 37. Inhan‑ Garip A, Aksu B, Akan Z, Akakin D, Ozaydin AN, San T. Eec ff t of extremely low frequency electromagnetic fields on growth rate and morphology of bacteria. Int J Radiat Biol. 2011;87(12):1155–61. 38. Amani S, Taheri M, Movahedi MM, Mohebi M, Nouri F, Mehdizadeh A. Evaluation of short‑term exposure to 2.4 GHz radiofrequency radia‑ tion emitted from Wi‑Fi routers on the antimicrobial susceptibility of Pseudomonas aeruginosa and Staphylococcus aureus. Galen Med J. 2020;9:1580. 39. Novickij V, Stanevičienė R, Vepštaitė‑Monstavičė I, Gruškienė R, Krivo ‑ rotova T, Sereikaitė J, Novickij J, Servienė E. Overcoming antimicrobial resistance in bacteria using bioactive magnetic nanoparticles and pulsed electromagnetic fields. Front Microbiol. 2018;9(8):2678. 40. Quinteros MA, Aristizábal VC, Dalmasso PR, Paraje MG, Páez PL. Oxida‑ tive stress generation of silver nanoparticles in three bacterial genera and its relationship with the antimicrobial activity. Toxicol In Vitro. 2016;1(36):216–23. 41. Pareek V, Gupta R, Panwar J. Do physico‑ chemical properties of silver nanoparticles decide their interaction with biological media and bacteri‑ cidal action? A review. Mater Sci Eng, C. 2018;1(90):739–49. 42. Akter M, Sikder MT, Rahman MM, Ullah AA, Hossain KF, Banik S, Hosokawa T, Saito T, Kurasaki M. A systematic review on silver nanoparticles‑induced cytotoxicity: Physicochemical properties and perspectives. J Adv Res. 2018;1(9):1–6. 43. Mourenza Á, Gil JA, Mateos LM, Letek M. Oxidative stress‑ generating antimicrobials, a novel strategy to overcome antibacterial resistance. Antioxidants. 2020;9(5):361. 44. Liu W, Worms I, Slaveykova VI. Interaction of silver nanoparticles with antioxidant enzymes. Environ Sci Nano. 2020;7(5):1507–17. 45. Loutfy SA, Al‑Ansary NA, Abdel‑ Ghani NT, Hamed AR, Mohamed MB, Craik JD, Eldin TA, Abdellah AM, Hussein Y, Hasanin MT, Elbehairi SE. Anti‑ proliferative activities of metallic nanoparticles in an in vitro breast cancer model. Asian Pac J Cancer Prev. 2015;16(14):6039–46. 46. Hegyi G, Szigeti GP, Szász A. Hyperthermia versus oncothermia: cellular effects in complementary cancer therapy. Evid‑Based Complement Altern Med. 2013;2013. 47. Simkó M, Mattsson MO. Extremely low frequency electromagnetic fields as effectors of cellular responses in vitro: possible immune cell activation. J Cell Biochem. 2004;93(1):83–92. 48. Schuermann D, Mevissen M. Manmade electromagnetic fields and oxida‑ tive stress—biological effects and consequences for health. Int J Mol Sci. 2021;22(7):3772. 49. Xu A, Wang Q, Lin T. Low‑frequency magnetic fields (LF‑MFs) inhibit Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : proliferation by triggering apoptosis and altering cell cycle distribution in breast cancer cells. Int J Mol Sci. 2020;21(8):2952. fast, convenient online submission 50. Khan MS, Alomari A, Tabrez S, Hassan I, Wahab R, Bhat SA, Alafaleq NO, thorough peer review by experienced researchers in your field Altwaijry N, Shaik GM, Zaidi SK, Nouh W. Anticancer potential of biogenic silver nanoparticles: a mechanistic study. Pharmaceutics. 2021;13(5):707. rapid publication on acceptance 51. Zielinska E, Tukaj C, Radomski MW, Inkielewicz‑Stepniak I. Molecular support for research data, including large and complex data types mechanism of silver nanoparticles‑induced human osteoblast cell death: • gold Open Access which fosters wider collaboration and increased citations protective effect of inducible nitric oxide synthase inhibitor. PLoS ONE. 2016;11(10):e0164137. maximum visibility for your research: over 100M website views per year 52. Brzóska K, Grądzka I, Kruszewski M. Silver, gold, and iron oxide nanoparti‑ cles alter miRNA expression but do not affect DNA methylation in HepG2 At BMC, research is always in progress. cells. Materials. 2019;12(7):1038. Learn more biomedcentral.com/submissions 53. Lin B, Williams‑Skipp C, Tao Y, Schleicher MS, Cano LL, Duke RC, Scheinman RI. 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Anticancer and antibacterial potentials induced post short-term exposure to electromagnetic field and silver nanoparticles and related pathological and genetic alterations: in vitro study

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10.1186/s13027-022-00416-4
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Abstract

Background: Resistance to antibiotics and anticancer therapy is a serious global health threat particularly in immu‑ nosuppressed cancer patients. Current study aimed to estimate the antibacterial and anticancer potentials of short‑ term exposure to extremely low frequency electromagnetic field (ELF‑EMF) and silver nanoparticles (AgNPs) either in sole or combined form. Methods: Antibacterial activity was evaluated via determination of the bacterial viable count reduction percentage following exposure, whereas their ability to induce apoptosis in breast cancer (MCF‑7) cell line was detected using annexin V‑fluorescein isothiocyanate and cell cycle analysis. Also, oxidative stress potential and molecular profile were investigated. Results: ELF‑EMF and AgNPs significantly (p < 0.01) reduced K. pneumonia viable count of compared to that of S. aureus in a time dependent manner till reaching 100% inhibition when ELF‑EMF was applied in combination to 10 µM/ml AgNPs for 2 h. Apoptosis induction was obvious following exposure to either ELF‑EMF or AgNPs, however their apoptotic potential was intensified when applied in combination recording significantly (p < 0.001) induced apoptosis as indicated by elevated level of MCF‑7 cells in the Pre G1 phase compared to control. S phase arrest and accumulation of cells in G2/M phase was observed following exposure to AgNPs and EMF, respectively. Up‑regulation in the expression level of p53, iNOS and NF‑kB genes as well as down‑regulation of Bcl‑2 and miRNA‑125b genes were detected post treatment. Conclusions: The antibacterial and anticancer potentials of these agents might be related to their ability to induce oxidative stress, suggesting their potentials as novel candidates for controlling infections and triggering cancer cells towards self‑ destruction. *Correspondence: Shbel.rania@gmail.com Microbiology and Immunology Department, Faculty of Pharmacy, Ahram Canadian University (ACU), 4th Industrial Zone, Banks Complex, 6th October City, Cairo, Egypt Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 2 of 16 Keywords: Antibacterial, Anticancer, Electromagnetic field, Silver nanoparticles, Cell cycle, Apoptosis, Oxidative stress Introduction the differential electrical behavior between the cancer The progressing emergence and the rapid spread of bac - and the normal cells. This signaling plays a key role in terial resistance to antibiotics is considered an alarming blocking cellular functions leading to induction of pro- world-wide health problem which strengthens the need grammed cell death [9]. Recently, a study reported that for alternative therapies. This continually growing prob - the tumor suppressive effects of the ELF‐EMF could pre - lem of antibiotic resistance not only endangers the public sent a new approach for the treatment of breast cancer if health, but also endures a massive negative impact on the this technology is clinically applied [10]. It was demon- economic development due to delayed hospitalization strated that the ability of pulsed low-frequency EMF to and recovery time in addition to the need for expensive modify the membrane integrity of cancer cells presents medications as well as specialized care for patients [1]. a new strategy in anticancer therapy. These pulsed mag - Many researchers have directed their efforts to manage netic fields (PMF) could selectively destruct the cancer the problem of antibiotic resistance via estimating the cell membranes without the use of ionizing radiation or effectiveness of new antibacterial agents either alone or cytotoxic agents. Thus, these fields could be applied as in combinations [2]. In the same context, breast cancer is adjuvants in cancer therapy to facilitate the delivery of considered the second common leading cause of cancer anticancer agents to tumor cells [11]. It was also found death among women [3]. Although many cases of cancer that EMF enhanced the in vivo anti-tumor efficacy of cis - initially respond to chemotherapy, but resistance is usu- platin against Lewis lung carcinoma cells [12]. Moreover, ally developed later [4] in addition to the undesirable side the exposure of glioblastoma brain cancer cells to a com- effects that are associated with the currently available bination between EMF and the anticancer agent temozo- chemotherapeutic drugs. Thus, there is also an urgent lomide enhanced the apoptosis via elevated expression demand for developing biocompatible and cost-effective of P53, Bax, and Caspase-3 (pro-apoptotic) genes while anticancer agents [5]. decreasing the expression levels of Bcl-2 and Cyclin-D1 Extremely low frequency electromagnetic field (ELF- (anti-apoptotic) genes [13]. EMF) is one of the most recent applications that exhib- Additionally, the recent advances in nanotechnology ited significant interactions with the living cells. However, offered new horizons in nanomedicine, facilitating the the mechanism of this interaction is still not clarified. synthesis of nanoparticles (NPs) that could be applied as Recent studies were carried out to assess the biological powerful weapon against pathogenic bacteria and cancer influence of such fields on different types of living cells cells [14]. especially on bacterial cells. Multi-directional alterations Among nanotechnology-based therapeutics, AgNPs following bacterial exposure to ELF-EMF were reported attracted the attention of many researchers due to such as ultra-structural and growth kinetics changes their distinctive characteristics and marked therapeutic [6]. Whereas, other studies found that ELF-EMF could potential in treating different diseases [14]. The antimi - enhance or suppress bacterial functional parameters. crobial activities of AgNPs either alone [15] or in com- Therefore, investigating the influence of ELF-EMF on posites with polymer [16] have been demonstrated in bacteria is essential not only for evaluating the impact of addition to their anticancer [17] as well as their antian- environmental stress on biological systems, but also to giogenic potentials [18]. AgNPs are now considered a explore the possibility of using the ELF-EMF to control valuable and non-traditional alternative to antibiotics the resistance to antibiotics [7]. with high antimicrobial potential against multidrug- ELF-EMF generating medical devices were also applied resistant (MDR) Gram-positive and Gram-negative for treatment of cancer patients in intensive care units. bacterial pathogens [19]. It was also reported that Consequently, during the outgoing 25  years research- AgNPs inhibited the proliferation of human glioblas- ers tried to investigate the impact of exposure to ELF- toma cells [20] as well as human breast cancer (MCF- EMF on cellular and molecular behavior in addition to 7) cells [21]. It was also found that AgNPs stimulated its effect on cancer cell metabolism. It was found that pro-apoptotic genes leading to interference with nor- exposure to 50/60  Hz magnetic field  promoted changes mal cellular functions and induction of programmed in signal transduction pathways that were directly associ- cell death. A study reported that AgNPs induced apop- ated to proliferative processes [8]. A study reported that tosis in NIH3T3 fibroblast cells is mediated via gen - electromagnetic field (EMF) could selectively hinder the eration of reactive oxygen species (ROS) and activation oxidation–reduction signaling in cancer cells relying on of Jun N-terminal kinases (JNK) pathway leading to M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 3 of 16 mitochondrial dependent apoptosis [22]. Recently, the Cell culture microenvironmental influence of titanium dioxide NPs Breast cancer (MCF-7) cells (HTB-22) were kindly sup- as a mechanical stimulus on cancer cells has been also plied from the International Center for Training and observed [23]. In addition to the reported concentra- Advanced Researches (ICTAR-Egypt). Cells were cul- tion dependent metabolic disturbing effect of graphene tured in RPMI-1640 media (Hyclone-USA), supple- oxide nanosheets on MCF-7 cells [24]. This alteration mented with 10% heat inactivated fetal bovine serum in the metabolomic profiling of cancer cells could con - (FBS), 100 U/ml penicillin and 100  μg/ml streptomycin. trol many malignant properties that are responsible for Cells were incubated in a humidified atmosphere of 5% tumorigenesis [25]. It was also found that the mecha- CO at 37 °C (Jouan-France) till reaching confluency. nobiological studies of AgNPs in cancer metabolomics suggested that AgNPs might be promising tools that Characterization of commercial AgNPs could be explored to develop enhanced anticancer ther- AgNPs were purchased as a commercial product by apy [26]. Nawah scientific (Cairo-Egypt). Characterization of the Consequently, the present study aimed to evaluate the particle size distribution and surface charge of the pre- antibacterial and the anticancer potentials of short-term pared particles were determined using Zetasizer Nano- exposure to ELF-EMF and AgNPs either in sole or com- ZS (Malvern Instruments-UK). Morphology and mean bined form at different time intervals. The mechanism size of AgNPs were  determined using Field Emission of action of these agents were elucidated via estimating Scanning Electron Microscopy (FESEM) (JSM-7600F, their ability to induce oxidative stress and their effects Joel-Japan), at accelerating voltages of 15  kV as previ- on the antioxidant enzymes. Apoptosis induction abil- ously described by Alkawareek et al. [27]. ity in MCF-7 cells was examined using different staining techniques as well as cell cycle analysis. Monitoring the Antibacterial potential expression profiles of five genes namely p53, inducible Determination of minimal inhibitory concentration nitric oxide synthase (iNOS) and nuclear factor-kappaB (MIC) of the tested AgNPs against S. aureus and K. (NF-kB), B cell lymphoma-2 (Bcl-2) and microRNA-125b pnemonia was carried out using broth micro dilution (miR-125b) were also investigated following treatment. method according to the CLSI reference standards [28]. One hundred microliter of Muller-Hinton broth (MHB) (Oxoid-UK) were distributed in 96 multi-well microtiter Materials and methods plates (TPP-Swiss). Double fold serial dilutions of AgNPs Bacterial strains and culture conditions were performed. Bacterial inoculum was prepared by Clinical isolate of Staphylococcus aureus (S. aureus) and adjusting the OD at 600  nm of the previously prepared Klebsiella pneumonia (K. pneumonia) was used as a bacterial suspension to reach a turbidity of 0.5 McFarland model for Gram-positive and Gram-negative bacteria, standard which is equivalent to 10   CFU/ml. The pre - respectively. Both isolates were obtained from Kasr Al- pared suspension was further diluted and inoculated in Ainy Teaching Hospital. S. aureus was isolated from pus all plates at a final concentration of 5 × 10  CFU/ml. Posi- specimen and identified using Gram stain, biochemical tive and negative control in each plate were considered. catalase and coagulase tests in addition to formation of Plates were incubated at 37  °C for 24  h and examined golden yellow colonies on nutrient agar as well as man- visually against dark background for absence or pres- nitol fermentation on mannitol salt agar. K. pneumonia ence of turbidity. The MIC was determined as the lowest was recovered from urine specimen and the isolate was concentration of AgNPs with no visible bacterial growth cultured on blood agar and MacConkey’s agar. Colonies compared to control. Twenty µl from each well with no were identified as Klebsiella pneumonia by biochemical observed bacterial growth were further inoculated on the reaction and confirmation was carried out using the API- surface of  agar plates and incubated overnight at 37 °C. 20E (BioMérieux-France) test system according to the The lowest concentration of AgNPs that kills > 99.9% of manufacturer’s instructions. the initial bacterial inoculum is considered as the mini- The bacterial concentration of each isolate was adjusted mum bactericidal concentration (MBC). to an optical density (OD)  of 0.1 at 600  nm which is equivalent to 10 colony forming units/ml (CFU/ml) and Electromagnetic field treatment inoculated on nutrient agar plate (pH of 7.0 ± 0.2) fol- Fresh subcultures of S. aureus and K. pneumonia at a lowed by incubation for 24 h at 37 °C. Before performing final concentration of 5X10   CFU/ml were aliquoted each experiment, three colonies from each isolate  were in sterile polystyrene plastic screw capped tubes and collected from each agar plate and inoculated in 5  mL treated with different concentrations of AgNPs (2.5, 5 & nutrient broth to obtain fresh subcultures. Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 4 of 16 Hematoxylin and eosin staining 10 µM/ml) at 37 °C for 1 & 2 h interval. In the meantime, Fifty micro liters of ELF-EMF treated MCF-7 cells either another set of tubes containing the same concentrations in presence or in absence of AgNPs were dispensed on of bacteria and AgNPs were exposed to ELF-EMF of 1 m clean slides (3 slides for each treatment). Slides were Tesla for the same time interval at 37 °C. Whereas, other air-dried, methanol fixed and rehydrated using descend - tubes inoculated with the same concentration of bacteria ing concentrations of alcohol (100%, 90%, 75% and 50%). were treated with ELF-EMF in absence of AgNPs. AgNPs Slides were washed with distilled water for 5  min. The or ELF-EMF untreated bacterial suspensions were con- slides were immersed in filtered hematoxylin stain for sidered as positive  control. At the end of the treatment 3 min and washed with distilled water twice followed by period, samples were obtained from all tubes to deter- immersion in filtered eosin stain for 5 s and washed with mine the bacterial viable count. The percentage reduc - distilled water. Dried slides were immersed in xylene fol- tion in the viable count was calculated for all examined lowed by mounting with Canada balsam. The coverslips samples compared to control. were mounted to each slide and left to air dry. Micro- MCF-7 cells pre-cultured 75 cm tissue culture flasks scopic fields (100X) were photographed using digital (SPL-Korea) were dissociated using 0.25% trypsin–EDTA camera (Canon-Japan), connected to a light microscope. (Lonza-Swiss) post decanting the exhausted growth The photomicrographs were evaluated for the presence media. Detached cells were cold centrifuged (Jouan, Ki- of morphological features of apoptosis [30]. 22-France),  phosphate buffer saline (PBS) washed and resuspended in 20  ml RPMI-1640 serum free media. Apoptosis detection Cells were equally aliquoted in sterile polystyrene tubes, ELF-EMF treated MCF-7 cell suspension, mixed with treated with AgNPs I C value and exposed to ELF-EMF and without AgNPs were quantitatively examined for of 1 m Tesla for 1 and 2 h interval. Similar concentrations detection of apoptosis using annexin V-fluorescein iso - of MCF-7 cells were treated with ELF-EMF at the same thiocyanate (FITC) apoptosis detection kit (Trevigen- conditions but in absence of AgNPs. Untreated control USA). Briefly, treated and untreated cells were PBS cells were also considered. ELF-EMF treated cells either washed and collected by centrifugation. Cells were dark in presence or absence of AgNPs were examined for incubated for 15  min at room temperature with 100  μl pathological changes in addition to cell cycle and molec- annexin-V incubation reagent: 10 µl (10 X) binding buffer ular analysis as well as biochemical tests. 1, 10 µl propidium iodide (PI), 1 µl annexin V-FITC and 79 µl de-ionized water. Samples were treated with 400 μl Cytotoxicity binding buffer (1 X) and analyzed using flow cytometer Cytotoxic effect of different concentrations of AgNPs was within 1 h for maximal signal. determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyl-tetrazolium bromide (MTT) assay, where growth Cell cycle analysis media were  decanted from 96-well micro titer plates ELF-EMF treated MCF-7 cell suspension, mixed with pre-cultured with  MCF-7 cells. AgNPs were applied in and without AgNPs were processed for cell cycle analy- double fold serial dilutions to MCF-7 precultured plates. sis. Cell cycle distribution was examined by measuring Untreated wells served as negative control and plates the DNA content of nuclei labeled with propidium iodide were incubated at 37  °C for 24  h. Post incubation, the (PI). Cell suspensions were collected by cold centrifuga- plates were washed three times with PBS as 250  μl/well. tion, washed with 1 ml cold PBS, centrifuged, and fixed in Fifty μl of MTT solution (0.5 mg/ml) were added to each 70% cold ethanol (Sigma Aldrich-USA) at + 4 °C for 24 h. well and plates were incubated for further 4  h at 37  °C. Cells were re-suspended in PBS containing 40  μg/ml PI, Plates were PBS washed three times and the formed blue 0.1 mg/ml RNase and 0.1% (v/v) Triton X-100. Post dark colored formazan was dissolved using 50  μl/well DMSO incubation at 37  °C for 30  min, the cells were analyzed (Sigma Aldrich-USA) followed by shaking the plates for using flow cytometer (Becton–Dickinson, San Jose, CA, 10  min at room temperature. Optical density (OD) was USA) equipped with an argon ion laser at a wavelength measured at 570  nm using ELISA plate reader (Biotek, of 488  nm. The cell cycle and sub-G1 group were deter - ELX-800-USA). The percentage of cellular viability was mined and analyzed. calculated, and the half maximal inhibitory concentration (IC ) was determined as the concentration resulting in Real time‑PCR 50% inhibition of cellular growth following 24 h exposure Total RNA was extracted from control, 1 and 2  h ELF- to AgNPs compared to the untreated control cells using EMF treated MCF-7 cells in presence and in absence of GraphPad prism software version 5 (S. Diego-USA) [29]. AgNPs using RNeasy Mini Kit (Qiagen-USA) accord- ing to manufacturer’s instructions. The concentration M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 5 of 16 and purity of the extracted RNA was evaluated using determined via evaluation of the percentage inhibition Beckman dual spectrophotometer (Beckman-USA). rate colorimetrically. The expression level of apoptosis-related genes; P53 (F: 5’-TCA GAT CCT AGC GTC GAG CCC-3’ & R: 5’-GGG Catalase (CAT) TGT GGA ATC AAC CCA CAG-3’), BCL-2 (F: 5’-GTG Catalase enzyme activity was evaluated using an AAC TGG GGG AGG ATT GT-3’& R: 5’-GGA GAA Amplex red catalase assay kit (Molecular Probes Inc.). ATC AAA CAG AGG CC-3’), NF-kB (F: 5’-CGC ATC The principle of the assay depends on that Amplex Red CAG ACC AAC AAC A-3 & R: 5’-TGC CAG AGT TTC reagent reacts with H O in the presence of horserad- 2 2 GGT TCA C-3’), iNOS (F: 5’-AGT ATG CAA TGA ATG ish peroxidase (HRP) and catalase enzyme to produce GGG AA-3’ & R: 5’-ATT CGA TAG CTT GAG GTA highly fluorescent resorufin. The assay was carried out GA-3’), miR-125b (F: 5’-ACT GAT AAA TCC CTG AGA according to the manufacturer’s instructions. Briefly, CCC TAA C-3’ & R: 5’-TAT GGT TGT TCT GCT CTC catalase containing samples as well as control samples TGT CAC-3’)   and β-actin (F: 5’-AGA GCT ACG AGC were diluted in reaction buffer and transferred to 96-well TGC CTG AC-3’ & R: 5’-AGC ACT GTG TTG GCG microplate (25  μl/well). Hydrogen peroxide prepared as TAC AG-3’) were determined using real-time PCR. Ten 40 μM H O was added to each well (25  μl/well). The 2 2 nanograms of the extracted total RNA from each sample reaction mixture was incubated for 30 min at room tem- were used for cDNA synthesis using high capacity cDNA perature followed by adding 50 μl/well Amplex Red/HRP. reverse transcriptase kit (Thermo Fischer Scientific- The plate was incubated for 30  min at 37  °C protected USA). The obtained cDNA was subsequently amplified from light. The fluorescence intensity was evaluated in using Sybr Green I PCR master kit (Thermo Fisher Sci - all wells by reading the plate using an excitation range of entific Inc.- Lithuania) using StepOne apparatus (Applied 530–560 nm and emission at 590 nm. The activity of cat - Biosystems-Thermo Fischer Scientific), as follows: 10 min alase enzyme was determined by subtracting the sample at 95  °C for enzyme activation followed by 40 cycles value from that of negative control. of 15  s at 95  °C, 20  s at 55  °C and 30  s at 72  °C for the amplification step. Changes in the expression of the tar - Statistical analysis get genes were normalized relative to the mean critical All experiments were performed in three independ- threshold (CT) values of β-actin as a housekeeping gene. ent tests. Data were presented as the mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) and Tukey post-hoc test. Statisti- Biochemical analysis cal analysis was carried out using statistical package Assessment of reactive oxygen species, superoxide dis- for social sciences (SPSS) software (version 25), IBM, mutase and catalase levels were determined in bacterial USA. Results were considered statistically significant at models as well as in MCF-7 cancer cell model following probability < 0.05. exposure to either ELF-EMF or AgNPs in addition to the combined treatment at different time interval. Bacterial models were treated with 5 µM/ml AgNPs, while MCF-7 Results cells were treated with IC of AgNPs. Antibacterial potential Recorded data revealed that AgNPs showed greater inhibitory potentials on K. pneumonia compared to that Reactive oxygen species (ROS) in case of S. aureus, where the recorded MICs were in the Assessment of the generated reactive oxygen species was order of 3.125 and 12.5  µM/ml, respectively. It was also determined according to the manufacturer’s protocol observed that the recorded MICs exerted bactericidal using ELISA kit, Catalog No. K936-100. activity on more than 99.9% of the initial bacterial inocu- lum, so the recorded MICs were considered as MBCs. Results showed that S. aureus viable count reduction Superoxide dismutase (SOD) percentage post exposure to sole EMF for 1 and 2 h time SOD was evaluated using PromoKine kit-Catalog Num- interval was 20.4% ± 3.4 and 28.5% ± 0.9, respectively. ber: PK-CA577-K335 (PromoCell-Germany) accord- At  the same time, AgNPs exhibited a statistically sig- ing to the manufacturer’s instruction. The assay is nificant (P < 0.05) concentration dependent reduction in based on that superoxide dismutase is an antioxidative S. aureus recording 4.1% ± 0.8, 10.3% ± 1.5& 15.5% ± 3.5 enzyme which catalyzes the dismutation of the super- post 1  h exposure to 2.5, 5 & 10  µM/ml AgNPs, respec- oxide anion into hydrogen peroxide and molecular oxy- tively. Extension of the treatment period with AgNPs for gen. The rate of the reduction with a superoxide anion 2 h resulted in higher reduction in the bacterial count by is inhibited by SOD. Therefore, activity of SOD could be Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 6 of 16 recording 43.9% ± 9.7, 50% ± 5.4 and 57.3% ± 4.6, respec- 9.6% ± 1.7, 17.7% ± 2.8 and 21.8% ± 5.6, respectively. An tively. A higher reduction in the bacterial count was enhancement in the antibacterial activity was recorded observed when AgNPs were applied in combination to when AgNPs were applied in combination to EMF, EMF where they recorded 66.5% ± 12.3, 81.3% ± 1.5 and where the percentage reduction recorded 31.6% ± 2.2, 92.6% ± 7.4 post 1 h treatment, respectively. In the same 35.8% ± 4.6 & 42.3% ± 1.6 and 45.2% ± 8.3, 53.1% ± 2.5 context, maximum inhibition was recorded following 2 h and 59.4% ± 3.6 following 1 and 2  h treatment interval, exposure to 2.5 and 5  µM/ml AgNPs in combination to respectively (Fig. 1a). EMF in the order of 87.4% ± 2.3 and 97.2% ± 5.5, respec- Similar pattern of antibacterial activity was observed tively, till achieving 100% inhibition when EMF was in case of K. pneumonia but with statistically signifi - applied to the bacterial suspension in presence of 10 µM/ cant (p < 0.01) higher inhibitory potential compared to ml AgNPs (Fig. 1b). S. aureus. Treatment of K. pneumonia with EMF for 1 and 2  h resulted in reduction in  the bacterial count by 30.1% ± 2.9 and 41.3% ± 1.4, respectively. K. pneumonia Biochemical analysis in bacterial models viable count was reduced in a concentration depend- Evaluation of the ROS and SOD in S. aureus following ent manner in the order of 33.2% ± 5.3, 40.5% ± 1.8& 1 h exposure to EMF, AgNPs and EMF + AgNPs revealed 45.4% ± 5.6 post 1  h exposure to 2.5, 5 and 10  µM/ml elevated ROS levels by 0.8-, 1.0- and 1.52-fold as well as AgNPs, respectively. The inhibitory potentials were an increase in SOD by 1.12-, 0.8- and 1.98-fold compared increased by extending the incubation period to 2  h Fig. 1 1a: Evaluation of antibacterial activity of sole EMF and AgNPs as well as EMF combined with AgNPs on S. aureus viable count at 1 and 2 h time interval. 1b: Assessment of the percentage reduction of K. pneumonia viable count post 1 and 2 h exposure to EMF, AgNPs and EMF in combination to AgNPs. *P < 0.001, **P < 0.01, ***P < 0.05 M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 7 of 16 Fig. 2 a Mean fold change in ROS, SOD and CAT in S. aureus 1 and 2 h post treatment with EMF, AgNPs and EMF + AgNPs compared to control. b Biochemical analysis of ROS, SOD and CAT levels following K. pneumonia exposure to EMF, AgNPs and EMF + AgNPs at 1 and 2 h time interval. **P < 0.01, ***P < 0.05 to control, respectively. Further incubation for 2  h was accompanied by higher ROS and SOD levels in the order of 1.48-, 1.23- and 2.2-fold for ROS and 1.69-, 1.72- and 2.44-fold in case of SOD, respectively (Fig.  2a). K. pneu- monia also showed elevated levels of ROS by a value of 1.2-, 1.3- and 1.68-fold as well as 1.69-, 1.42- and 2.86- fold 1 and 2  h following exposure, respectively. In addi- tion to increased SOD levels by values of 1.17-, 1.58- and 2.0-fold as well as 1.73-, 1.58- and 2.52-fold 1 and 2  h post exposure, respectively (Fig. 2b). On the contrary, treatment of S. aureus with EMF, AgNPs and EMF + AgNPs for 1  h showed a marked Fig. 3 Evaluation of cellular viability of MCF‑7 cells post 24 h treatment with AgNPs using MTT assay indicating elevated viability reduction in CAT levels by 0.9-, 1.21- and 2.04-fold as along with decreasing AgNPs concentration. ***P < 0.05 well as 2.16-, 2.14- and 2.88-fold reduction post 2 h expo- sure (Fig.  2a). Comparable reduced levels of CAT were detected in K. pneumonia post 1  h treatment recording of apoptosis such as shrinkage of cells and peripheral 2-, 2.12- and 2.43-fold reduction as well as reduced CAT condensation of chromatin. Necrotic swollen cells with levels by 2.36-, 2.91- and 4.23-fold post 2  h treatment mixed euchromatin and heterochromatin and ruptured compared to control, respectively (Fig. 2b). cell membrane were also observed (Fig.  4e). Apoptotic shrunken cells with shrunken nuclei, peripheral con- Cytotoxicity densation of chromatin and irregular cell membranes The cytotoxic effect of AgNPs 24  h post MCF-7 cells were detected post 1 and 2  h treatment with AgNPs, treatment was determined using MTT assay. Recorded respectively (Fig.  4b, f ). Treatment with ELF-EMF in data revealed that viability was concentration dependent, combination to AgNPs for 1  h revealed the presence where the  viability increases as long as the concentra- of swollen necrotic cells and swollen nuclei with mixed tions of AgNPs decrease till reaching 100% viability at a euchromatin and heterochromatin and ruptured cell concentration of 1  μM/ml. The calculated IC value of membranes. Shrunken apoptotic cells with peripheral AgNPs was 4.15 ± 0.20 μM/ml (Fig. 3). condensation of chromatin in addition to secondary necrotic cells with peripheral condensation of chroma- Hematoxylin and eosin staining tin and ruptured cell membranes as well as apoptotic Microscopic examination of MCF-7 cells treated with bodies were also observed (Fig.  4c). Necrotic cells with sole ELF-EMF for 1  h revealed the detection of swol- mixed euchromatin and heterochromatin, ruptured cell len cells, swollen nuclei with mixed euchromatin and membranes, intranuclear eosinophilic structures as well heterochromatin, ruptured cell membranes as well as as shrunken apoptotic cells with irregular cellular and intranuclear eosinophilic structures (Fig. 4a). Extending nuclear membranes were detected post 2 h exposure to the exposure time to 2 h showed characteristic features ELF-EMF in combination to AgNPs (Fig.  4g). On the Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 8 of 16 Fig. 4 Pathological changes detected post MCF‑7 cells exposure to sole ELF‑EMF, AgNPs and ELF‑EMF in combination to AgNPs at different time interval using hematoxylin and eosin staining. a MCF‑7 cells photomicrograph post 1 h exposure to ELF‑EMF showing swollen cells, swollen nuclei with mixed euchromatin and heterochromatin (Yellow arrows) as well as ruptured cell membranes (Green arrows) in addition to intranuclear eosinophilic structures (Red arrows). e Photomicrographs following 2 h treatment with ELF‑EMF revealing the occurrence of apoptosis as indicated by the observed shrunken apoptotic cells with peripheral condensation of chromatin (Green arrows) as well as necrotic cells with mixed euchromatin and heterochromatin (Yellow arrows) and swollen cell with ruptured cell membrane (Red arrow). b&f Photomicrographs of MCF‑7 cells post exposure to AgNPs for 1 and 2 h, respectively revealing the presence of shrunken apoptotic cells with shrunken nuclei (Yellow arrows), peripheral condensation of chromatin (Green arrows) and irregular cell membranes (Red arrows). c Swollen necrotic MCF‑7 cells and swollen nuclei with mixed euchromatin and heterochromatin and ruptured cell membranes (Blue arrows) were detected post 1 h exposure to ELF‑EMF in combination to AgNPs. Shrunken apoptotic cell (Green arrow) with peripheral condensation of chromatin (Orange arrow) in addition to secondary necrotic cells with peripheral condensation of chromatin and ruptured cell membranes (yellow arrow) as well as apoptotic bodies (Red arrows) were also observed. g Photomicrograph post 2 h exposure to ELF‑EMF in combination to AgNPs showing necrotic cells with mixed euchromatin and heterochromatin (Red arrows), ruptured cell membranes (Green arrows), intranuclear eosinophilic structures (Yellow arrows) and shrunken apoptotic cells with irregular cell and nuclear membranes (Black arrows). d&h Control untreated MCF‑7 cells examined at 1 and 2 h, respectively showing regular cells with hyperchromatic nuclei (Original magnification 100X, Oil) contrary, untreated cells showed regular appearance 8.26% ± 1.0) and AgNPs (10.45% ± 4.2 and 7.65% ± 0.7), with hyperchromatic nuclei, respectively (Fig. 4d, h). respectively (Fig. 5). Apoptosis Cell cycle analysis Recorded data showed that the sole exposure to Recorded data revealed a time dependent apoptosis ELF-EMF and AgNPs or in combination signifi - as indicated by the elevation of MCF-7 cells in Pre G1 cantly (P < 0.001) induced time dependent apopto- phase (16.85% ± 2, 24.11% ± 3.1) post treatment with sis compared to untreated cells. Also, a statistically ELF-EMF for 1 and 2  h, respectively. Treatment with significant (P < 0.01) induction of apoptosis post 1  h AgNPs resulted in a similar pattern of apoptosis induc- exposure to ELF-EMF-AgNPs combined form record- tion recording 14.34% ± 5.5, 20.35% ± 0.8 of cells in Pre ing 16.65% ± 1.3 and 11.48% ± 1.3 of the analyzed cells G1 phase post 1 and 2  h exposure, respectively. In the in late apoptosis and necrosis, respectively, compared same context, MCF-7 cell treatment with ELF-EMF in to lower levels of the detected cells (5.61% ± 1.8 and combination to AgNPs induced a statistically significant 5.87% ± 2.1) post treatment with sole ELF-EMF or (p < 0.001) time dependent higher levels of apoptotic AgNPs (7.82% ± 1.1 and 6.29% ± 3.8), respectively. Fur- cells (22.45% ± 3.5, 38.26% ± 5.4) post 1 and 2  h treat- ther exposure to ELF-EMF combined with AgNPs for ment, respectively. Also, sole treatment with ELF-EMF 2  h significantly (P < 0.01) resulted in a marked eleva- induced a significant (p < 0.01) time dependent G2/M tion of early apoptotic and necrotic cells in the order phase arrest (24.52% ± 2.2, 35.53% ± 1.8) compared to of 21.84% ± 2.5 and 13.71% ± 1.8 compared to that in untreated cells (18.95% ± 1.5, 20.76% ± 3.2). Whereas case of treatment with sole ELF-EMF (11.59% ± 1.5 and significant (p < 0.01) arrest during S phase (40.68% ± 7.1, 51.24% ± 2.2) was observed 1 and 2  h post exposure M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 9 of 16 Fig. 5 Evaluation of early and late apoptosis as well as necrosis post MCF‑7 cells post exposure to ELF‑EMF, AgNPs and ELF‑EMF/AgNPs for 1 and 2 h interval using annexin V‑FITC apoptosis detection kit. *P < 0.001, **P < 0.01, ***P < 0.05. a necrotic cells, b late apoptosis, c early apoptosis to sole AgNPs, respectively. Similarly, combined treat- observed post 1 h exposure to combined treatment com- ment with ELF-EMF and AgNPs resulted in a significant pared to that recorded 2 h post treatment recording 0.42- (p < 0.05) S phase arrest (49.43% ± 4.6, 55.17% ± 3.1) 1 and 0.55-fold change, respectively (Fig. 7). and 2 h post treatment, respectively (Fig. 6). Biochemical analysis in MCF‑7 cells The oxidative stress induced 1 h post exposure of MCF-7 Gene expression profile cells to ELF-EMF, AgNPs and ELF-EMF combined with Expression level of apoptosis related genes was evaluated, AgNPs showed statistically significant (P < 0.05) elevated it was found that p53, iNOS and NF-kB genes showed levels of the generated ROS recording 1.18- 1.0- and 2.04- significant up-regulation recording 4.86-, 3.47- and 5.98- fold compared to untreated cell control, respectively. fold increase post 1  h exposure compared to 6.6-, 4.43- Whereas, extending the exposure time to 2 h resulted in and 7.37-fold elevation post 2  h treatment, respectively. increased levels of ROS in the order of 1.45-, 1.72- and Down-regulation in the expression level of Bcl-2 and 2.88-fold compared to untreated cell control, respectively. miRNA-125b in the order of 0.69- and 0.68-fold was Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 10 of 16 Fig. 6 Evaluation of cell cycle profile post short ‑term exposure of MCF‑7 cells to ELF‑EMF and AgNPs as sole treatment as well as in combination at 1 and 2 h time interval using flow cytometry. *P < 0.001, **P < 0.01, ***P < 0.05 Fig. 7 Evaluation of the expression profile of apoptosis related genes in MCF‑7 cells post sole exposure to ELF‑EMF and AgNPs as well as in combination at different time interval using real time PCR. **P < 0.01, ***P < 0.05 Recorded data showed a significant (P < 0.05) elevated Discussion SOD levels recording 1.55-, 1.43- and 2.33-fold increase Multi-drug resistance is a threatening obstacle in the 1 h post treatment with ELF-EMF, AgNPs and ELF-EMF/ treatment of infectious diseases, where the misuse of AgNPs, respectively. While MCF-7 cells following 2  h broad-spectrum antibiotics has evoked antibiotic resist- treatment exhibited higher enhancement of SOD activ- ance among several human bacterial pathogens. AgNPs ity in the order of 2.06-, 1.8- and 2.43-fold compared to as well as ELF-EMF have attracted much attention in this untreated cells, respectively. On the other side, the activ- field due to their recorded antibacterial potentials [6]. ity of catalase was significantly (P < 0.05) reduced post 1 h u Th s, the present study tried to highlight the antibacte - treatment with ELF-EMF, AgNPs and ELF-EMF/AgNPs rial potentials developed following exposure to these two by 2.04-, 1.17- and 2.66-fold, respectively. In the same weapons either alone or in combination. For this issue, context, the activity of catalase was also reduced post 2 h S. aureus and K. pneumonia were selected as models for treatment by 2.23-, 2.0- and 4.0- fold, respectively (Fig. 8). Gram-positive and Gram-negative bacteria, respectively. S. aureus is a commensal bacterium known to asymp- tomatically colonize the human skin, nasal passages, and gastrointestinal tract. S. aureus infections range from mild skin and soft tissue infections to more severe M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 11 of 16 Fig. 8 Assessment of the generation of ROS, SOD and CAT post MCF‑7 cells exposure to ELF‑EMF and AgNPs as sole treatment as well as in combination at 1 and 2 h time interval. **P < 0.01, ***P < 0.05 invasive diseases, such as endocarditis, bacteremia, sep- cytoplasm as well as degrading the chromosomal DNA sis, pneumonia, and osteomyelitis [31]. In the meantime, which subsequently result in failure of ATP produc- K. pneumonia accounts for a significant proportion of tion and chromosomal replication [33]. Another study hospital-acquired urinary tract infections, pneumonia, examined E. coli post exposure to AgNPs using trans- septicemia as well as soft tissue infections and it has mission electron microscope to observe the interaction become multi-resistant to various types of antibiotics. between them. It was found that the positively charged k. pneumonia infections are also of clinical importance Ag ions were attracted to the negatively charged bac- among patients in intensive care units with compromised terial lipopolysaccharides, induce the formation of immune systems such as cancer patients [6]. The present holes in the bacterial cell wall and result in cell lysis study aimed also to investigate the influence of short- [34]. It is important to note that the rate of dissolution term exposure to ELF-EMF and AgNPs either in sole of AgNPs to release Ag + ions is multifactorial, where it or combined form at different time intervals. Thus, we is dependent on several factors including their physico- selected 1 and 2 h time interval as a model for the short- chemical properties such as the size, shape, concentra- term exposure time. tion, capping agent and colloidal state of NPs as well as Similar to the current findings it was reported that the presence of chlorine, thiols, sulfur, and oxygen [35]. AgNPs exhibited antibacterial activity against E. coli In the current study, the recorded MICs and the in a concentration and time dependent manner at a viable count following treatment with AgNPs revealed range of low concentrations in the order of 10  µM and that these particles showed greater inhibitory poten- 100 µM [32]. Many other studies demonstrated that the tials towards Gram-negative bacteria compared to that antibacterial potential of AgNPs isn’t only specific for observed in case of Gram-positive bacteria. Similar Gram-negative bacteria (E. coli, K. pneumonia and P. findings were reported in another study, where they aeruginosa), however its activity also extents to Gram demonstrated higher antibacterial activity of AgNPs positive (S. aureus and B. subtilis) bacterial strains. It against E. coli compared to that exerted on S. aureus was reported that the cytotoxic effect of AgNPs against [36]. That was attributed to the structural differences in bacteria may result from the oxidative dissolution of the composition of the cell walls between Gram-posi- AgNPs and the release of Ag ions from AgNPs. The tive and Gram-negative bacteria. Gram-negative bacte- released Ag ions interact with sulfhydryl (-SH) groups ria have an outer layer of lipopolysaccharides and thin of cell wall-bound enzymes and proteins, then inter- layer of peptidoglycan which confers for the overall lack fere with the respiratory chain of bacteria resulting in of rigidity of the cell wall of Gram-negative bacteria. disruption of the bacterial cell wall. In addition to the On the other side, Gram-positive bacteria have a three- ability of the released ions to penetrate the bacterial dimensional rigid cell wall consisting of thicker layer of cell wall and react with thiol groups of the proteins in peptidoglycan which is formed of linear polysaccharide Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 12 of 16 chains cross-linked by short peptides. The rigidity and due to the production of ROS. It was reported that the cross-linking not only diminish the cell wall attachment positively charged silver ions released from AgNPs could sites for AgNPs in Gram-positive bacteria but also induce oxidative stress in bacterial cells due to their increase the difficulty of AgNPs penetration to the bac - interference with the normal function of the bacterial terial cell wall [19]. electron transport chain and thus facilitating the genera- In the same context to the antibacterial potentials of tion of ROS. ROS generation is primarily responsible for AgNPs, other studies demonstrated the bacterial inhibi- the bacterial death as it enhances lipid peroxidation but tory activity of ELF-EMF. It was found that exposure of hindered ATP production and DNA replication [40]. At different Gram-positive and Gram-negative bacterial the same time, another study attributed the antibacte- strains to ELF-EMF resulted in reduction in their growth rial potential of AgNPs to the combined effect of these rates with respect to control samples. In addition to the particles on the bacterial components as previously dem- ultrastructural changes that were observed post treat- onstrated in addition to their ability to induce oxidative ment with this type of electromagnetic waves [37]. Also, stress [41]. another study reported a reduction in the percentage It is important to point out that, SOD and CAT are viability of S. aureus and E. coli post exposure to ELF- antioxidant enzymes which have been identified as criti - EMF. That was justified by the ability of these waves to cal modulators in AgNPs induced oxidative stress and alter the structure and function of the ion channels and are considered as bioindicators of increased ROS pro- efflux pumps of bacterial cell walls resulting in an altera - duction. [42]. AgNPs induced oxidative stress in bacte- tion in the permeability of the bacterial cell wall to dif- rial cells results in the generation of superoxide anion •− ferent molecules leading to cell death. Although many (O ). This free radical could be dismuted to hydrogen researchers tried to explore the effect of EMF on bacte -peroxide (H O ) by the effect of SOD enzyme. The pro - 2 2 ria, but these effects are variable depending on the fre -duced H O is then quickly converted to H O and O by 2 2 2 2 quency and intensity of EMF, exposure time as well as CAT enzyme. The H O produced by the effect of SOD 2 2 the phase of bacterial growth, ingredients of the media, could penetrate the bacterial membranes and interacts 2+ genetic properties, presence or absence of oxygen, and with ferrous ion (F e ) and thiol groups (-SH) of protein bacterial membrane features [38]. In accordance with the cysteines leading to inactivation of essential enzymes of 2+ current findings, it was demonstrated that the antibacte - the pathogen. Moreover, F e is oxidized during a Fenton rial potentials of either AgNPs or EMF were highly effec - reaction by H O and generates hydroxyl radical, which 2 2 tive against Gram-negative bacteria compared to that in turn causes further destruction in the bacterial pro- recorded in case of Gram-positive [2]. teins, DNA, and lipids [43]. In agreement with the present study, it was reported Similar to our results, a study found that the activity of that EMF is a versatile tool which could be successfully SOD in AgNPs treated P. aeruginosa was elevated, how- used for increasing the susceptibility of bacteria to anti- ever the activity of CAT was reduced post treatment. bacterial agents. They reported elevated antibacterial This study reported that AgNPs enhanced the activity of potentials when iron NPs were applied in combination to SOD enzyme resulting in an accumulation of H O . On 2 2 EMF against Bacillus subtilis and E. coli bacterial mod- the other hand, AgNPs treated cells failed to get rid of els. They attributed this elevated inactivation efficiency to the accumulated H O due to suppression of CAT activ- 2 2 the induction of higher local field gradients, hyperther - ity by the effect of AgNPs. Accordingly, AgNPs treated mia, and motion of both the bacterial cells and magnetic bacterial cells showed elevated levels of cell death due to NPs. Despite the ability of EMF to enhance the antibac- the enhancement of oxidative stress by the effect of the terial potentials of different antimicrobials such as NPs, accumulated H O [34]. That was suggested to be related 2 2 but their exact mechanism of action is still unclear and to the formation of AgNPs-CAT complex which in turn requires further investigations [39]. Thus, the present resulted in conformational changes in CAT enzyme study evaluated the levels of ROS as well as the antioxi- leading to an impairment of its enzymatic activity. In dant enzymes (SOD and CAT) to explore the reason for contrast, the formation of AgNPs-SOD complex has no the bacterial inhibitory potentials of AgNPs and ELF- influence on its enzymatic activity [44]. EMF either alone or in combination. Current findings Regarding the anticancer potential of AgNPs and in revealed that the antibacterial potential of AgNPs against agreement with the present findings, it was reported S. aureus and K. pneumonia was directly proportional that AgNPs reduced MCF-7 cellular viability in a to the detected elevated levels of ROS in both bacterial dose  dependent manner recording an IC value of models post treatment. Consequently, the recorded bac- 6.28 μM [45]. It was reported that excessive production terial inhibitory potentials could be justified by the abil - of ROS in cells by direct interaction with AgNPs and/ ity of AgNPs to induce oxidative stress in bacterial cells or dissolved silver ions is currently accepted as one of M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 13 of 16 the main mechanisms of cellular toxicity of engineered PI staining. Also, a significant time dependent elevated nanoparticles in living organisms. Although ROS have late apoptosis was detected  following cellular treatment many signaling and information functions, but it could with EMF-AgNPs combination compared to  that  in also diminish the antioxidant defense system leading case of sole application of EMF and AgNPs as well as to to damage of DNA, lipids and proteins [44]. That was untreated cell control. obvious in the current study, where the recorded results Cell cycle analysis in addition to  the assessment of the revealed a time dependent reduction in CAT enzyme gene expression profile of 5 apoptosis related genes were activity, which is considered an antioxidant enzyme, also performed to explore the correlation between the post treatment with AgNPs either alone or in combi- detected apoptosis and the influence of the applied treat - nation to EMF. The observed CAT enzymatic activity ments on cell cycle phases as well as the gene expression reduction was also in parallel to apoptosis induction pattern. Recorded results proved the potential of EMF, following treatment with AgNPs as well as production AgNPs and  their combination to induce apoptosis as of ROS. This might be attributed to AgNPs induced they significantly induced elevated level of cells in Pre G1 generation of oxidative stress which was intensified by phase. The exposure to AgNPs was also accompanied by reduction of CAT enzymatic activity resulting in H O S phase arrest. On the other hand, following the exposure 2 2 accumulation as previously explained. to EMF there was a significant elevation in the percent - The recorded ability of EMF to induce apoptosis in age of cells in G2/M phase. In agreement with the current MCF-7 cells could be related to some reports that  high- findings, it was reported that treatment of MCF-7 cells lighted the ability of electromagnetic field to induce with an I C concentration of AgNPs generated progres- hyperthermia in tumor cells. It was demonstrated that sive accumulation of cells in the S phase of the cell cycle hyperthermia could kill cancer cells but with limited haz- [45]. Another study reported the accumulation of MCF-7 ards on healthy cells due to the well-known biophysical cells in the G2/M phase 6, 12 and 24  h following expo- differences between the cancer cells and their healthy sure to EMF [49]. counterparts. Hyperthermia not only induce apoptosis Regarding investigating the mechanism of action of in cancer cells but may also enhance the susceptibility of the EMF and AgNPs on apoptosis at the molecular level. cancer cells to anticancer agents thus allowing the reduc- The expression levels of two pro-apoptotic genes (p53 tion of their administered doses [46]. That was obvious in and iNOS) as well as two anti-apoptotic genes (Bcl-2 and the present findings, where the current results reported miRNA-125b) in addition to the impact on the expres- a time dependent ability of the combination between sion level of NF-kB were evaluated following exposure EMF and AgNPs to enhance apoptosis in AgNPs treated to different treatments. In agreement with the current MCF-7 cells compared to the lower apoptosis induced in findings, a recent study reported that the expression lev - case of sole application of AgNPs. els of pro-apoptotic genes (p53, Bax and caspase-3) were In accordance with the current findings, a study inves - significantly up-regulated, whereas the expression of the tigated the biochemical consequences following exposure anti-apoptotic gene Bcl-2 was significantly down-regu - to EMF and suggested that the genotoxic events associ- lated in AgNPs treated MCF-7 and colon cancer (HCT- ated with exposure to EMF might be due to its ability 116) cells [50]. to elevate the levels of free radicals which in turn led to The current study aimed to explore other novel mecha - DNA damage [47]. Another in  vivo study demonstrated nisms that might be involved in AgNPs or EMF induced alteration in different oxidative stress biomarkers (SOD cytotoxicity on MCF-7 cells. Thus, the expression profile and CAT) as well as increased ROS levels in the brain of of iNOS gene was investigated. Similar reports revealed male rats following exposure to ELF-EMF for 2 h. It was that AgNPs elevated the expression level of iNOS genes also found that the exposure of murine squamous cell as well as nitric oxide (NO) derived reactive species in carcinoma line (AT478) to ELF-EMF for 16 min resulted human osteoblast cells [51]. However, it is essential to in an increased ROS formation and SOD activities. Simi- prove the generation of NO in our study on the transi- lar observations showed elevated ROS formation and tional level not only on the molecular level. In accordance induced cell death post 1  h exposure of breast cancer with the current findings, a study demonstrated down- (MDA-MB-231) cells to EMF [48]. regulation in the expression levels of various miRNA The ability of AgNPs and EMF to induce apoptosis genes following treatment of cancer liver (HepG2) cells either alone or in combination was proved in the pre- with different types of nanoparticles. The reported sent study using several techniques. For example, hema- changes were highly induced by AgNPs followed by gold toxylin and eosin staining indicated the enhancement of and iron nanoparticles and at  the same time they were apoptosis in MCF-7 cells. These results were confirmed accompanied by inhibition of both cellular proliferation via flowcytometric analysis using annexin V-FITC and and tumorigenesis [52]. Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 14 of 16 It is worth to point out that NF-kB gene plays a dual exposure time. The recorded reduction in the bacterial role in apoptosis, where it could function as both a pro- viable count following exposure to these agents was apoptotic and anti-apoptotic regulatory factor within higher against Gram-negative bacteria as compared a single cell type [53]. It was also reported that the pro- to Gram-positive bacterial model. These antibacterial apoptotic or anti-apoptotic function of NF-kB is deter- potentials were suggested to be related to the capa- mined by the nature of the apoptotic stimulus. u Th s, bility of these agents to induce oxidative stress by the the nature of the signals evoked by the respective death generation of ROS. However, their effect was magnified enhancers determines whether NF-kB induction leads via enhancing the antioxidant activity of SOD and on to apoptosis or survival, suggesting that the modulation the other side reducing the activity of catalase enzyme of NF-kB activity may present a new approach in can- resulting in elevated toxicity that might be attributed cer adjuvanted therapy [54]. A study demonstrated that to H O accumulation. Consequently, the combination 2 2 the NF-kB pathway has been proposed to be a key fac- between the tested agents could present a novel strat- tor contributing to the unusual phenotype and aggres- egy for infection control and to overcome bacterial siveness of breast cancer. In agreement with our results, resistance. In the meantime, a time dependent induc- it was reported that some up-regulated NF-kB-related tion of apoptosis was observed following treatment of genes could serve as novel therapeutic targets in breast MCF-7 cells with AgNPs, ELF-EMF as well as in com- cancer [55]. bination. That was proposed to the ability of the tested To the best of our knowledge, this is the first study that treatments to significantly elevated the Pre G1 apop - tried to explore the apoptosis induction potentials of the totic phase of MCF-7 cells. Moreover, the exposure to combination between ELF-EMF and AgNPs. Also, the AgNPs induced S phase arrest, whereas the EMF treat- mechanisms that may be involved in the antibacterial and ment was accompanied by accumulation of cells in the anticancer potentials of the short-term exposure to ELF- G2/M phase. Additionally, up-regulation in the expres- EMF in combination to AgNPs have not been previously sion level of p53, iNOS and NF-kB genes was observed, investigated. Current findings strongly suggest that the however down-regulation of the anti-apoptotic genes, ability of ELF-EMF in combination to AgNPs to induce namely Bcl-2 and miRNA-125b was detected post oxidative stress in bacterial and cancer cells via genera- treatment. Biochemical analysis also shed light on the tion of ROS, SOD induction and catalase reduction could ability of both EMF and AgNPs to induce apoptosis via be responsible for their antibacterial and anticancer generation of oxidative stress. Finally, it could be con- potentials. The significance of this study isn’t only related cluded that AgNPs and ELF-EMF either in sole applica- to exploring the antibacterial and the anticancer poten- tion or in combination could be considered as potential tials of AgNPs and ELF-EMF either alone or in combi- oxidative stress generating agents that might pave the nation as new therapeutic approaches, but it spotlight way to solve the problem of antibiotic resistance espe- on the effectiveness of the combination between these cially in immunocompromised cancer patients and agents as an essential life-saving approach if this type of could successfully direct cancer cells to death. treatments could be applied clinically. That might offer greater health improvement especially in the immuno- Abbreviations compromised cancer patients who are more vulnerable AgNPs: Silver nanoparticles; Bcl‑2: B cell lymphoma‑2; CAT : Catalase; ELF‑EMF: to develop infections with antibiotic resistant pathogens. Extremely low frequency electromagnetic field; FITC: Fluorescein isothiocy‑ anate; iNOS: Inducible nitric oxide synthase; miR‑125b: MicroRNA‑125b; NF‑kB: Nuclear factor‑kappaB; PI: Propidium iodide; ROS: Reactive oxygen species; Study limitations SOD: Superoxide dismutase. This research study was performed on  only two kinds Acknowledgements of pathogenic bacterial models, one cancer cell model Not applicable and only at one tested intensity (1  m Tesla) of the EMF. Authors’ contributions Further studies on different bacterial and cancer cell AFM: conceptualization, methodology, resources, investigation, software, writ‑ models as well as different exposure conditions are ing‑ original draft. MN: resources, methodology, investigation, writing review. recommended. MEA: resources, methodology, investigation, writing review. TMMA: resources, methodology, investigation, writing review. WMA‑E: resources, methodology, investigation, writing review. HFK: resources, methodology, investigation, Conclusions writing review. EERM: resources, methodology, investigation, writing review. LEEM: resources, methodology, investigation, writing review. TAG: resources, AgNPs and ELF-EMF could be considered as poten- methodology, investigation, writing review. MAE‑FD: resources, methodology, tial antibacterial and anticancer agents. The activities investigation, writing review. RIS: conceptualization, methodology, resources, of these agents were enhanced upon their combina- investigation, software, writing‑ original draft, writing‑review, editing and submitting the paper to the journal. All authors read and approved the final tions in a time dependent manner even though at short manuscript. M ohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 15 of 16 Funding 12. Tofani S, Barone D, Berardelli M, Berno E, Cintorino M, Foglia L, Ossola P, This research received no specific grant from any funding agency in the pub ‑ Ronchetto F, Toso E, Eandi M. Static and ELF magnetic fields enhance the lic, commercial, or not‑for ‑profit sectors. in vivo anti‑tumor efficacy of cis‑platin against lewis lung carcinoma, but not of cyclophosphamide against B16 melanotic melanoma. Pharmacol Availability of data and materials Res. 2003;48(1):83–90. All data generated or analyzed during this study are included in this published 13. Akbarnejad Z, Eskandary H, Dini L, Vergallo C, Nematollahi‑Mahani SN, article. Farsinejad A, Abadi MF, Ahmadi M. Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low‑frequency electromagnetic field (100 Hz, 100 G). Declarations Biomed Pharmacother. 2017;1(92):254–64. 14. Sriram MI, Kanth SB, Kalishwaralal K, Gurunathan S. Antitumor activity Ethical approval and consent to participate of silver nanoparticles in Dalton’s lymphoma ascites tumor model. Int J Not applicable as the study didn’t include human participants. Nanomedicine. 2010;5(5):753–62. https:// doi. org/ 10. 2147/ IJN. S11727. PMID: 21042 421; PMCID: PMC29 62271. Consent for publication 15. Banerjee I, Pangule RC, Kane RS. Antifouling coatings: recent develop‑ Not applicable. ments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms. Adv Mater. 2011;23(6):690–718. Competing interests 16. Sanpui P, Murugadoss A, Prasad PD, Ghosh SS, Chattopadhyay A. The The authors declare that they have no competing interests. antibacterial properties of a novel chitosan–Ag‑nanoparticle composite. Int J Food Microbiol. 2008;124(2):142–6. Author details 17. Gomathi AC, Rajarathinam SX, Sadiq AM, Rajeshkumar S. Anticancer activ‑ International Center for Training and Advanced Researches (ICTAR‑Egypt), ity of silver nanoparticles synthesized using aqueous fruit shell extract of Cairo, Egypt. Histology Department, Faculty of Medicine, Al‑Azhar University, Tamarindus indica on MCF‑7 human breast cancer cell line. J Drug Deliv Cairo, Egypt. Histology Department, Faculty of Medicine, Al‑Azhar Univer ‑ Sci Technol. 2020;55:101376. sity, Damietta, Egypt. Pathology Department, Faculty of Medicine, Al‑Azhar 18. Gurunathan S, Lee KJ, Kalishwaralal K, Sheikpranbabu S, Vaidyanathan R, University, Cairo, Egypt. Microbiology and Immunology Department, Faculty Eom SH. Antiangiogenic properties of silver nanoparticles. Biomaterials. of Pharmacy, Ahram Canadian University (ACU), 4th Industrial Zone, Banks 2009;30(31):6341–50. Complex, 6th October City, Cairo, Egypt. 19. Franci G, Falanga A, Galdiero S, Palomba L, Rai M, Morelli G, Galdiero M. Silver nanoparticles as potential antibacterial agents. Molecules. Received: 26 October 2021 Accepted: 14 January 2022 2015;20(5):8856–74. 20. AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxic‑ ity and genotoxicity of silver nanoparticles in human cells. ACS Nano. 2009;3(2):279–90. 21. Franco‑Molina MA, Mendoza‑ Gamboa E, Sierra‑Rivera CA, Gómez‑Flores References RA, Zapata‑Benavides P, Castillo ‑ Tello P, Alcocer‑ González JM, Miranda‑ 1. Malik B, Bhattacharyya S. Antibiotic drug‑resistance as a complex system Hernández DF, Tamez‑ Guerra RS, Rodríguez‑Padilla C. Antitumor activity driven by socio‑ economic growth and antibiotic misuse. Sci Rep. of colloidal silver on MCF‑7 human breast cancer cells. J Exp Clin Cancer 2019;9(1):9788. Res. 2010;29(1):1–7. 2. El‑Kaliuoby MI, Khalil AM, El‑Khatib AM, Shehata N. Antibacterial syner ‑ 22. Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ. The apoptotic effect gism of electrospun nanofiber mats functioned with silver nanoparticles of nanosilver is mediated by a ROS‑and JNK ‑ dependent mechanism and pulsed electromagnetic waves. Polymers. 2021;13(2):277. involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett. 3. Chan K, Morris GJ. Chemoprevention of breast cancer for women at high 2008;179(3):130–9. risk. Semin Oncol. 2006;33(6):642–6. https:// doi. org/ 10. 1053/j. semin oncol. 23. Raja G, Cao S, Kim DH, Kim TJ. Mechanoregulation of titanium dioxide 2006. 08. 017 (PMID: 17145342). nanoparticles in cancer therapy. Mater Sci Eng C. 2020;107:110303. 4. Johnston SR. Acquired tamoxifen resistance in human breast can‑ 24. Raja G, Selvaraj V, Suk M, Suk KT, Kim TJ. Metabolic phenotyping analysis cer–potential mechanisms and clinical implications. Anticancer Drugs. of graphene oxide nanosheets exposures in breast cancer cells: metabo‑ 1997;8(10):911–30. lomics profiling techniques. Process Biochem. 2021;1(104):39–45. 5. Brown K. Breast cancer chemoprevention: risk‑benefit effects of the 25. Raja G, Jung Y, Jung SH, Kim TJ. 1H‑NMR‑based metabolomics for cancer antioestrogen tamoxifen. Expert Opin Drug Saf. 2002;1(3):253–67. targeting and metabolic engineering–a review. Process Biochem. 2020. 6. El‑kaliuoby MI, Khalil AM, El‑Khatib AM, Shalaby TI. Synergistic antibacte ‑ 26. Raja G, Jang YK, Suh JS, Kim HS, Ahn SH, Kim TJ. Microcellular environ‑ rial effect of silver nanoparticles and extremely low‑frequency pulsed mental regulation of silver nanoparticles in cancer therapy: a critical magnetic fields on klebsiella pneumoniae. Communications. 2018;15:23. review. Cancers. 2020;12(3):664. 7. Segatore B, Setacci D, Bennato F, Cardigno R, Amicosante G, Iorio R. 27. Alkawareek MY, Bahlool A, Abulateefeh SR, Alkilany AM. Synergistic Evaluations of the effects of extremely low‑frequency electromagnetic antibacterial activity of silver nanoparticles and hydrogen peroxide. PLoS fields on growth and antibiotic susceptibility of escherichia coli and ONE. 2019;14(8):e0220575. pseudomonas aeruginosa. Int J Microbiol. 2012;2012:587293. https:// doi. 28. Wayne PA, Clinical and laboratory standards institute. Performance stand‑ org/ 10. 1155/ 2012/ 587293. ards for antimicrobial susceptibility testing. twenty‑first informational 8. Falone S, Santini S Jr, Cordone V, Di Emidio G, Tatone C, Cacchio M, Ami‑ supplement. M100–S21. CLSI. 2011: 100–121. carelli F. Extremely low‑frequency magnetic fields and redox ‑responsive 29. Vijayakumar S, Ganesan S. In vitro cytotoxicity assay on gold nanoparti‑ pathways linked to cancer drug resistance: insights from co‑ exposure‑ cles with different stabilizing agents. J Nanomater. 2012. https:// doi. org/ based in vitro studies. Front Public Health. 2018;23(6):33. 10. 1155/ 2012/ 734398 9. Tofani S. Electromagnetic field exposure system for the study of possible 30. Sholqamy MI, Abd‑ElHamid ES, Mostafa AH, Mohamed AF, El‑Said WA. anti‑ cancer activity. IEEE Trans Electromagn Compat. 2002;44(1):148–51. Monitoring the anticancer effects of two different gold nanostructures 10. Oh IR, Raymundo B, Jung SA, Kim HJ, Park JK, Kim CW. Extremely low‑ shapes towards Hep‑2 Cells. Int J Med Nano Res. 2019;6:028. https:// doi. frequency electromagnetic field altered PPARγ and CCL2 levels and sup ‑ org/ 10. 23937/ 2378‑ 3664. 14100 28. pressed CD44+/CD24− breast cancer cells characteristics. Bull Korean 31. Jenkins A, Diep BA, Mai TT, Vo NH, Warrener P, Suzich J, Stover CK, Chem Soc. 2020;41(8):812–23. Sellman BR. Differential expression and roles of Staphylococcus 11. Ashdown CP, Johns SC, Aminov E, Unanian M, Connacher W, Friend J, Fus‑ aureus virulence determinants during colonization and disease. MBio. ter MM. Pulsed low‑frequency magnetic fields induce tumor membrane 2015;6(1):e02272‑ e2314. disruption and altered cell viability. Biophys J. 2020;118(7):1552–63. 32. Orlov IA, Sankova TP, Babich PS, Sosnin IM, Ilyechova EY, Kirilenko DA, Brunkov PN, Ataev GL, Romanov AE, Puchkova LV. New silver Mohamed et al. Infectious Agents and Cancer (2022) 17:4 Page 16 of 16 nanoparticles induce apoptosis‑like process in E. coli and interfere with antiapoptotic regulatory factor within a single cell type. Cell Death Differ. mammalian copper metabolism. Int J Nanomed. 2016;11:6561. 1999;6(6):570–82. 33. Liao C, Li Y, Tjong SC. Bactericidal and cytotoxic properties of silver nano‑ 54. Kaltschmidt B, Kaltschmidt C, Hofmann TG, Hehner SP, Dröge W, Schmitz particles. Int J Mol Sci. 2019;20(2):449. ML. The pro‑ or anti‑apoptotic function of NF‑κB is determined by the 34. Liao S, Zhang Y, Pan X, Zhu F, Jiang C, Liu Q, Cheng Z, Dai G, Wu G, Wang nature of the apoptotic stimulus. Eur J Biochem. 2000;267(12):3828–35. L, Chen L. Antibacterial activity and mechanism of silver nanoparticles 55. Lerebours F, Vacher S, Andrieu C, Espie M, Marty M, Lidereau R, Bieche I. against multidrug‑resistant Pseudomonas aeruginosa. Int J Nanomed. NF‑kappa B genes have a major role in inflammatory breast cancer. BMC 2019;14:1469. Cancer. 2008;8(1):1–1. 35. Ferdous Z, Nemmar A. Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure. Int J Publisher’s Note Mol Sci. 2020;21(7):2375. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 36. Schiffman JD, Wang Y, Giannelis EP, Elimelech M. Biocidal activity lished maps and institutional affiliations. of plasma modified electrospun polysulfone mats functionalized with polyethyleneimine‑ capped silver nanoparticles. Langmuir. 2011;27(21):13159–64. 37. Inhan‑ Garip A, Aksu B, Akan Z, Akakin D, Ozaydin AN, San T. Eec ff t of extremely low frequency electromagnetic fields on growth rate and morphology of bacteria. Int J Radiat Biol. 2011;87(12):1155–61. 38. Amani S, Taheri M, Movahedi MM, Mohebi M, Nouri F, Mehdizadeh A. Evaluation of short‑term exposure to 2.4 GHz radiofrequency radia‑ tion emitted from Wi‑Fi routers on the antimicrobial susceptibility of Pseudomonas aeruginosa and Staphylococcus aureus. Galen Med J. 2020;9:1580. 39. Novickij V, Stanevičienė R, Vepštaitė‑Monstavičė I, Gruškienė R, Krivo ‑ rotova T, Sereikaitė J, Novickij J, Servienė E. Overcoming antimicrobial resistance in bacteria using bioactive magnetic nanoparticles and pulsed electromagnetic fields. Front Microbiol. 2018;9(8):2678. 40. Quinteros MA, Aristizábal VC, Dalmasso PR, Paraje MG, Páez PL. Oxida‑ tive stress generation of silver nanoparticles in three bacterial genera and its relationship with the antimicrobial activity. Toxicol In Vitro. 2016;1(36):216–23. 41. Pareek V, Gupta R, Panwar J. Do physico‑ chemical properties of silver nanoparticles decide their interaction with biological media and bacteri‑ cidal action? A review. Mater Sci Eng, C. 2018;1(90):739–49. 42. Akter M, Sikder MT, Rahman MM, Ullah AA, Hossain KF, Banik S, Hosokawa T, Saito T, Kurasaki M. A systematic review on silver nanoparticles‑induced cytotoxicity: Physicochemical properties and perspectives. J Adv Res. 2018;1(9):1–6. 43. Mourenza Á, Gil JA, Mateos LM, Letek M. Oxidative stress‑ generating antimicrobials, a novel strategy to overcome antibacterial resistance. Antioxidants. 2020;9(5):361. 44. Liu W, Worms I, Slaveykova VI. Interaction of silver nanoparticles with antioxidant enzymes. Environ Sci Nano. 2020;7(5):1507–17. 45. Loutfy SA, Al‑Ansary NA, Abdel‑ Ghani NT, Hamed AR, Mohamed MB, Craik JD, Eldin TA, Abdellah AM, Hussein Y, Hasanin MT, Elbehairi SE. Anti‑ proliferative activities of metallic nanoparticles in an in vitro breast cancer model. Asian Pac J Cancer Prev. 2015;16(14):6039–46. 46. Hegyi G, Szigeti GP, Szász A. Hyperthermia versus oncothermia: cellular effects in complementary cancer therapy. Evid‑Based Complement Altern Med. 2013;2013. 47. Simkó M, Mattsson MO. Extremely low frequency electromagnetic fields as effectors of cellular responses in vitro: possible immune cell activation. J Cell Biochem. 2004;93(1):83–92. 48. Schuermann D, Mevissen M. Manmade electromagnetic fields and oxida‑ tive stress—biological effects and consequences for health. Int J Mol Sci. 2021;22(7):3772. 49. Xu A, Wang Q, Lin T. Low‑frequency magnetic fields (LF‑MFs) inhibit Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : proliferation by triggering apoptosis and altering cell cycle distribution in breast cancer cells. Int J Mol Sci. 2020;21(8):2952. fast, convenient online submission 50. Khan MS, Alomari A, Tabrez S, Hassan I, Wahab R, Bhat SA, Alafaleq NO, thorough peer review by experienced researchers in your field Altwaijry N, Shaik GM, Zaidi SK, Nouh W. Anticancer potential of biogenic silver nanoparticles: a mechanistic study. Pharmaceutics. 2021;13(5):707. rapid publication on acceptance 51. Zielinska E, Tukaj C, Radomski MW, Inkielewicz‑Stepniak I. Molecular support for research data, including large and complex data types mechanism of silver nanoparticles‑induced human osteoblast cell death: • gold Open Access which fosters wider collaboration and increased citations protective effect of inducible nitric oxide synthase inhibitor. PLoS ONE. 2016;11(10):e0164137. maximum visibility for your research: over 100M website views per year 52. Brzóska K, Grądzka I, Kruszewski M. Silver, gold, and iron oxide nanoparti‑ cles alter miRNA expression but do not affect DNA methylation in HepG2 At BMC, research is always in progress. cells. Materials. 2019;12(7):1038. Learn more biomedcentral.com/submissions 53. Lin B, Williams‑Skipp C, Tao Y, Schleicher MS, Cano LL, Duke RC, Scheinman RI. NF‑κB functions as both a proapoptotic and

Journal

Infectious Agents and CancerSpringer Journals

Published: Feb 4, 2022

Keywords: Antibacterial; Anticancer; Electromagnetic field; Silver nanoparticles; Cell cycle; Apoptosis; Oxidative stress

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