Cellular stress response to extremely low‐frequency electromagnetic fields (ELF‐EMF): An explanation for controversial effects of ELF‐EMF on apoptosis

1 INTRODUCTION

In modern world, electromagnetic fields (EMFs) have become an inseparable part of routine life. Numerous electric power-generating human-made devices are now producing EMFs which are overlaid on those of earth's magnetic field. EMFs are usually identified with a 50 or 60 Hz frequency and therefore are classified under the extremely low-frequency, non-ionizing span of electromagnetic spectrum.1 Due to these physical characteristics, ELF-EMFs are not capable of breaking molecular bond or inducing thermal effects on tissue. However, it is now proven that they can interact with human tissues and induce some weak electrical currents.2 In addition, it is not completely understood whether biological effects induced by EMFs are hazardous for human or environment. During last few decades, a number of studies have reported beneficial effects of ELF-EMFs in treatment of cancer both in vitro and in vivo.3-7 Despite this, the exact mechanism of these anti-neoplastic effects has not been confirmed yet.

So far, the most probable mechanism proposed for explaining anticancer effects of ELF-EMF is induction of apoptosis through upregulation of intracellular reactive oxygen species (ROS) which has also been confirmed by different experimental studies. In the study performed by Ding et al.,8 it was demonstrated that 24-h exposure to 60 Hz, 5 mT ELF-EMF could potentiate apoptosis induced by H2O2 in HL-60 leukaemia cell lines. Similarly, in the study performed by Jian et al.,9 exposure to an intermittent 100 Hz, 0.7 mT EMF significantly enhanced rate of apoptosis in human hepatoma cell lines pretreated with low-dose X-ray radiation. Kaszuba-Zwoinska et al.10 also showed that short-term exposure of human acute monocytic leukaemia cell line exposure to 50 Hz, 45 ± 5 mT pulsed EMF, significantly potentiated rate of apoptosis induced by cyclophosphamide and colchicine. Benassi et al. reported that co-treatment of human ovarian adenocarcinoma cell lines with cisplatin, a chemotherapeutic agent with DNA-damaging and ROS-promoting activity, significantly enhanced sensitivity to apoptosis through increasing both caspases 3 and 9 activity. This is in accordance with previous studies demonstrating an enhancement in 1-methyl-4-phenylpyridinium (MPP +) induced caspase-dependent apoptosis following 24-h exposure to 50 Hz, 1 mT ELF-MF in SH-SY5Y neuroblastoma cell lines.11

One of the main mechanisms proposed for defining anticancer effects of ELF-EMF is induction of apoptosis through upregulation of reactive oxygen species (ROS) which has also been confirmed by different experimental studies.

Contrary to above-mentioned studies, several reports propose an anti-apoptotic activity for ELF-EMF. Pirozzoli et al.12 reported that 24-h exposure to 50 Hz, 1 mT ELF field significantly attenuated apoptosis induced by camptothecin in LAN-5 neuroblastoma cell lines. De Nicola et al reported that puromycin-induced apoptosis in human lymphoblasts was significantly weakened in response to 2-h exposure to a 0.1 mT ELF field.13 They reported that reduced glutathione (GSH) was the key mediator of the observed effect. In addition, based on the study performed by Palumbo et al.,14 pretreatment of Jurkat leukaemic cell lines with 50 Hz, 1 mT EMF resulted in 22% reduction in caspase 3-dependent apoptosis induced by anti-Fas therapy. Moreover, based on Cid et al.,15 the anti-apoptotic activity of melatonin on HepG2 cell lines was completely abrogated in response to 42-h intermittent exposure with a 50 Hz, 10 µT EMF. Similarly, bleomycin-induced apoptotic activity in K562 erythroleukaemia cell line was significantly reduced in response to a short-term (~10 min) exposure period to a 217 Hz, 120 µT ELF-MF.16 Based on Brisdelli et al.,17 concurrent treatment of K562 cell lines with ELF-EMF and quercetin for 24 h significantly increased expression of Bcl2, a protein with anti-apoptotic activity, compared with quercetin alone treated and control groups. They also reported that extending ELF-EMF exposure for 1–3 days results in attenuation of growth inhibitory effects of quercetin in leukaemia cell lines which was in association with reduced level of caspase 3 activity, along with inhibition of quercetin induced reduction in expression of Bcl-xL and Mcl-1 anti-apoptotic proteins.

Still, some reports have stated no statistically significant cytotoxic or cytostatic activity for ELF-EMF. Laqué-Rupérez et al.18 reported no statistically significant changes in methotrexate-induced cytotoxicity in MCF-7 breast cancer cell lines after exposing them to 25 Hz, 1.5 mT pulsed EMF. Similarly, in the study performed by Mizuno et al.,19 no statistically significant changes in survival rates of SV40 cells were observed between cells which were subjected to UV radiation alone and group subjected to concurrent administration of 24-h 60 Hz, 5 mT EMF and UV radiation. Finally, Höytö et al.20 reported no statistically significant enhancement in anti-proliferative and cytotoxic activities of menadione on SH-SY5Y neuroblastoma cells when combined with 24 h exposure to ELF-MF of 100 µT intensity.

This discrepancy in observations has made it difficult to come into a unit conclusion, and therefore, application of ELF-EMF in clinic for treatment of cancer still remains a big dilemma. In our idea, the main point neglected in interpreting the discrepancies observed in results is consideration of cellular stress responses induced by ELF-EMF exposure and its interplay with the molecular mechanisms underlying apoptosis. The main purpose of current review was to outline the triangle of ELF-EMF, cellular stress response of cells and apoptosis, and interpret and unify the discrepancies in results based on this theory. Therefore, initially we will explain studies performed on identifying the effect of ELF-EMF on induction/inhibition of apoptosis, enumerate proposed pathways through which ELF-EMF exposure may affect apoptosis; then, we will explain cellular stress response, cues for activation of this phenomenon in response to ELF-EMF exposure and finally under a separate “discussion” section we will try to explain why such controversy has been obtained by different investigators.

2 APOPTOSIS AND ELF-EMF EXPOSURE

Considering hallmarks of cancer, aberrant cellular survival is an important characteristic of malignant cells which is usually attributed to a mis-regulated apoptotic state in cells. Apoptosis is a type of programmed cell death which is abundantly observed under both physiological and pathological conditions, upon interaction of cells with specific stimulators, capable of activating either of intrinsic and extrinsic pathways. Moreover, failure in induction of apoptosis, as a consequence of aberrant expression of antigens, secreted angiogenic growth factors, or their receptors has already been linked to an elevated risk of metastasis, promotion of angiogenesis and an accelerated risk of resistance development to anti-angiogenic cancer therapies.21-27 Either mediated by the extrinsic (mediated by FASL, TNFα and so on) or intrinsic pathway (most importantly, accumulation of ROS and development of oxidative stress), the rest of the process will be followed by modulation of specific sets of procaspase molecules cleavage (caspase 8 and caspase 9 for extrinsic and intrinsic pathways respectively), ending in degradation of numerous intracellular target proteins, blebbing of cellular membrane, cleavage and degradation of chromosomal DNA, and finally, getting phagocytosed and scavenged by polymorphonuclear cells.28 Apoptosis can be triggered upon activation of two main pathways which are broadly referred as “intrinsic” and “extrinsic” pathways.

The most prevalent mechanism through which several chemotherapeutic agents trigger apoptosis is induction of mitochondrial membrane permeabilization, the intrinsic apoptosis pathway, which is mainly controlled by Bcl-2 proteins family. This process results in leakage of several pro-apoptotic molecules such as cytochrome c, Smac/DIABLO, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) into the cytoplasm.29 Released Endo G and AIF initiate nuclear modifications while the others activate caspases. Cytochrome c promotes formation of apoptosis protease activating factor-1 (Apaf-1) oligomers using ATP or dATP.30, 31 This complex in next place recruits procaspase 9 and forms “apoptosome” which in turn induces autoactivation of procaspase 9.32, 33 Matured caspase 9 further activates caspase 3 and 7 which in turn results in initiation of downstream caspase cascades34 and induction of apoptotic cell death. In parallel, Smac/DIABLO antagonize suppressing effects of inhibitors of apoptosis proteins (IAPs) on activated caspases.35, 36

In some cell types however, chemotherapeutic-induced apoptotic cell death may be initiated through the death receptor Fas (APO-1/CD95), the extrinsic apoptosis pathway. Ligation of Fas with its natural ligand, FasL, promotes Fas clustering, which in next place attracts FADD37 and procaspase 8,38 totally forming a complex referred as death-inducing signalling complex (DISC). The mature caspase 8 would be exhausted from the DISC after oligomerization and​autoactivation of procaspase 8.39 Based on the cell type, mature caspase 8 initiates apoptosis by two distinct pathways.40 In first pathway, high quantities of mature caspase 8 induce direct cleavage and activation of procaspase 3 without enrolment of mitochondrial pathway. In second pathway however, low quantities of mature caspase 8 are formed which is not capable of directly inducing activation of procaspase 3. Alternatively, herein, caspase 8 promotes cleavage of the “BH3-only protein” Bid and formation of truncated Bid which, in turn, triggers mitochondrial apoptosis pathway.41, 42

Different groups of anticancer agents are capable of activating death receptor pathway through enhancement of Fas or FasL expression.43 This process is transcription-dependent and involves p53 activity.44 The activated signalling pathway following Fas/FasL complexation outlines an autocrine/paracrine pathway like that happening during activation-induced cell death in T lymphocytes. Nevertheless, FasL plays minimal role in chemotherapy-induced apoptosis, as administration of antagonist antibodies or any small molecule preventing from FasL/Fas interaction does not suppress apoptosis.45 Likewise, the pro-apoptotic effects of chemotherapeutic agents on embryonic fibroblasts from FADD and caspase 8 knockout mice remained unaltered.46, 47

Although apoptosis is usually induced upon overproduction of ROS and development of oxidative stress, a mild-to-moderate level of ROS is required for maintenance and regulation of physiological function of cells including growth, proliferation, differentiation and migration52; regulation of immune system's function and maintaining redox balance48; and promotion of autophagy through activation of different signalling pathways including phosphoinositide 3-kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPK), nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1), nuclear factor-κB (NF-κB) and the tumour suppressor p53.48-51 Hence, manipulation of ROS level in cells is a good strategy for cancer therapy.

If ROS generation and accumulation can be considered the first cellular event of ELF-EMFs exposure, the modification of intracellular Ca2+ levels could be one of the most important mechanisms by which ROS have their multiple actions in cells.52 Over the past few years, lots of data have shown that ELF-EMF exposure regulates intracellular Ca2+ level which can, in turn, activate multiple physiological mechanisms such as differentiation of chromaffin cells into neuronal-like cells (ELF-MF, 60 Hz, 0.7 mT, 2 h/day twice a day53); cell death by apoptosis (ELF-MF, 50/60 Hz, 0.2–5 mT, 2–3 consecutive days54, 55); functional modification of the immune system's cells through involvement of P2Y membrane receptors (sinusoidal electric fields, 0.3 or 30 kV/m, 50 Hz, for 24 h56), activation of mechanically operated stretch-activated Ca2+ channels (noninvasive electrical stimulus, 0.1-V/cm direct current57); and the enhancement of the expression of voltage-gated Ca2+ channels in different human cell systems (static magnetic fields, 0.15 and 66 mT58). In this context, Kapri-Pardes et al. examined responses of cells (both transformed and non-transformed) to ELF-EMFs across a broad range of field strengths by examining activation of ERK1/2 and other signalling pathways. They reported that all cell lines could sense and respond to ELF-EMFs. Nevertheless, the extent to which transformed cells responded to EMFs was significantly lower compared to non-transformed ones, and interestingly, in MDA-MB-231 cells, exposure decreased phosphorylation of ERK1/2. Perhaps the more important finding of their study was that contrary to what previously was though, cells can sense magnetic field strengths as low as 0.15 µT which is at least partly mediated through activation of NADH oxidase.59

So far, multiple signalling pathways have identified to be affected under acute or short-term exposure to ELF-EMF. Indeed, exposure to ELF-EMF promotes tyrosine phosphorylation of specific protein components of signalling pathways in cells. For instance, it has been shown that 1- to 30-min exposure to 60 Hz, 0.1 mT ELF-EMFs results in activation of Lyn, a protein tyrosine kinase and serine/threonine kinase protein kinase C (PKC) in B lymphocytes.60 Likewise, acute exposure of Jurkat cell line (~5 min) to 50 Hz, 0.1 mT ELF-EMFs activates Lck, which in turn promotes complexation of T cell receptors.61 Similar result was also reported in adherent cells, where 5 min exposure to 50 Hz, 04 mT ELF-EMFs promoted epidermal growth factor receptor (EGFR) clustering and subsequent stimulation of Ras GTPases in long fibroblast cells of Chinese hamster.62 In addition, cyclic AMP/protein kinase A (cAMP/PKA) is another pathway which is activated in response to exposure to ELF-EMF in rat's cerebellar granule cells and human skin fibroblasts.63, 64

Mitogen-activated protein kinase (MAPK) cascades are among the other important signalling cascades which are stimulated upon exposure to ELF-EMF in several types of examined cells.65 MAPK pathways consisting from four main cascades, including extracellular signal regulated kinase 1 and 2 (ERK1/2), ERK5, p38 and c-Jun N-terminal kinase (JNK), are central in regulation of almost all stimulated cellular events such as differentiation, proliferation, stress responses and apoptosis.65, 66 After initial stimulation, these cascades function by serially activating specific protein kinases in each level of cascade with ultimate result of phosphorylation of thousands of target proteins and modulation of related cellular processes. Akt is another protein kinase with responsibilities similar to the MAPKs.67 Akt becomes activated in response to extracellular stimuli upon phosphorylation of its two activatory moieties following interaction with PI3K-phosphorylated phospholipids. Any dysregulation or abnormalities in mentioned five signalling pathways is associated with certain disorders including cancer.67, 68 Interestingly, both acute/short-term and chronic/long-term exposure to ELF-EMF has shown to induce activation of Akt and MAPK.69, 70 For instance, 30-min exposure to ELF-EMF results in activation of ERKs and Akt in several cancer cell lines including MCF7, HaCaT, NB69, HL-60 and so on.69-72 Furthermore, 3- to 15-min exposure to 50 Hz in CHL cells and 15, 30, or 60 min in NB69 cells results in activation of stress-associated MAPKs, p38 and JNK.70, 73, 74

In most of these studies, the strengths of applied ELF-EMFs were more than 100 µT, and none has investigated the relation between the changes in strength of EMFs and induction of cell signalling cascades. Recently, Kapri-Pardes et al. examined responses of cells (both transformed and non-transformed) to ELF-EMFs across a broad range of field strengths by examining activation of ERK1/2 and other signalling pathways. They reported that all cell lines could sense and respond to ELF-EMFs. However, the extent to which transformed cells responded to EMFs was significantly lower compared to non-transformed ones, and interestingly, in MDA-MB-231 cells, exposure decreased phosphorylation of ERK1/2. Perhaps the more important finding of their study was that contrary to what previously was though, cells can sense magnetic field strengths as low as 0.15 µT which is at least partly mediated through activation of NADH oxidase.59

Although effects of EMF exposure on TGF-β/BMP signalling pathway have been studied during the process of bone repair, same pathway is a key player in pathophysiology of cancer and its modulators demonstrate statistically significant anti-metastatic activities.75 Different studies have shown that exposure to pulsed EMF results in a statistically significant increase in TGF-β, in osteoblastic cells and both atrophic and non-hypertrophic cells.76, 77 In addition, based on a recent study, exposing differentiating osteoblasts to pulsed EMF, promotes activation of TGF-β signalling pathway through Smad2 and increases expression of osteoblastic differentiation markers such as ALP and type I collagen.78 BMP expression during osteogenesis was also increased after exposure to pulsed EMFs.79-81 Moreover, it has been shown that exposure to pulsed EMFs, stimulates osteogenic differentiation and maturation through the activation of BMP-Smad1/5/8 signalling. In this case, BMP receptor II, BMPRII, regulates differentiation in a cilium-dependent manner.82 Considering the separate effects of BMP and pulsed EMFs on differentiation and maturity of osteoblasts, many studies have shown that concurrent treatment with BMP and pulsed EMF enhances bone formation to a much greater degree compared to each treatment alone.83-86

In addition to the mentioned signalling pathways, electromagnetic fields can also affect pathways underlying VEGF and FGF signalling molecules.87, 88 Based on a recent report, exposure to pulsed EMF significantly increases expression of IGF-1 at mRNA level and promotes bone formation.89 In addition, pulsed EMF (1.5 mT, 75 Hz) can also increase synthesis of proteoglycans and protect human articular cartilage from further damage.90 Finally, it has been shown that exposure to pulsed EMF reverses osteoporotic effect of dexamethasone.91

Notch signalling is a highly conserved pathway that regulates cellular fate and skeletal development. Recent reports have shown that exposure to pulsed EMF can regulate expression levels of Notch4 receptor, as well as DLL4 ligands and target genes (Hey1, Hes1 and Hes5) during the osteogenic differentiation of human mesenchymal stem cells. Interestingly, expression of osteogenic markers, including Runx2, Dlx5, Osterix, Hes1 and Hes5, after pulsed EMF treatment was reversed following treatment of cells with notch pathway inhibitors.92 Furthermore, exposure to pulsed EMF significantly increases the level of cAMP, protein kinase A activity and accelerates osteogenic differentiation of MSCs.93, 94 Anti-inflammatory effects of pulsed EMFs have also been reported both in vitro95, 96 and in vivo,97-99 as well as in clinical settings.100

3 ELF-EMF AND INDUCTION OF CELLULAR STRESS RESPONSE

Numerous studies have shown that cells are physiologically well buffered against negative effects of ELF-EMF alone. However, in the presence of stressful condition, including exposure to toxins, viruses, DNA damage and proteotoxic, hypoxic, metabolic and oxidative stress, an additional weak stressor like ELF-EMF might produce large effects.101 Based on Mattsson and Simko who extensively investigated the oxidative response of cells following ELF-EMF exposure, ROS levels can be consistently altered in different cell types or experimental conditions following exposure to magnetic fields. These effects were prominent for fields with intensities more than 1 mT, but were also documented at or below 100 mT. Despite this, all observed effects where moderate and majority of changes were below 50%.102 Consequently, the produced amounts of ROS by ELF-EMF are not high enough to induce major DNA damage. Although this mild elevation in ROS levels in response to acute or chronic exposure to ELF-EMF cannot trigger cell death, it may induce cellular resistance against oxidative damage through upregulation of antioxidant pathways and induction of cellular stress response. Small change in ROS levels stated above is capable of promoting different cell signalling pathways especially by means of superoxide ions.20, 103-106 This phenomenon requires a certain time to develop and promotes several other time-dependent changes.105

As discussed earlier, antioxidant defence capacity of cells can be changed following exposure to ELF-EMF. For example, it has been shown that exposure to ELF-EMF can significantly increase SOD levels in cells.107 Furthermore, ELF-EMF can enhance activity of both glutathione-S-transferase and -reductase enzymes in malignant cells.108 Also, based on Cichon et al.,109 ELF-EMF exposure can upregulate expression of different antioxidant target genes including CAT, SOD1, SOD2, GPx1 and GPx4. In addition, multiple pathways involved in orchestrating cellular stress response of cells to stressful condition can also become activated following ELF-EMF exposure. Based on literature, generation of mitochondrial ROS at the time of ELF-EMF exposure is pivotal for activation of signalling pathways involved in cellular adaption.110 Activation and upregulation of Nrf2 expression, the master redox-sensing transcription factor may be the most prominent example in this regard which has been confirmed in a Huntington's disease-like rat model.111 Another cellular stress response to ELF-EMF involves activation of MAPK and NF-κB which, in turn, may upregulate expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and enhance mitochondrial biogenesis.112 Activation of autophagy, ER stress, heat-shock response and sirtuin 3 expression are among the other identified cellular stress responses to ELF-EMF exposure, all of which have been discussed earlier.

This cellular stress response is very important when ELF-EMF exposure is applied before chemotherapy. The main mechanism through which several chemotherapeutic agents induce apoptosis in cancer cells is elevation of ROS and induction of apoptosis. However, as antioxidant defence after ELF-EMF exposure is enhanced, cells become more resistant to these agents. Such effects have also been reported during radiation therapy and are responsible for development of resistance to radiotherapy. Contrarily, when chemotherapy and ELF-EMF exposure are performed simultaneously, this increase in ROS levels potentiates the oxidative stress induced by chemotherapeutic agents, as the ROS levels become excessively high and cells do not have time for adaption. Therefore, the result is enhancement of apoptosis. Differences between extent of apoptosis induced or when no significant differences are observed in combination are mostly dependent on the nature of the cell (ie the antioxidant defence), type and dose of the chemotherapeutic agent applied and the number of cells seeded in the plate.

A number of other harmful agents or conditions, such as thermal stress,113 exposure to alkylating agents,114 heavy metals115 and ionizing radiation116 have shown to initiate a similar response. Generally, cellular stress response is characterized by modulation of expression of various genes. The main outcome of this alteration in pattern of gene expression is protection of cells from cytotoxic doses of a harmful agent. This response represents that following exposure to a toxin, cells expect or at least prepare themselves for a lethal concentration of the agent. In addition to mild exposure to toxic agents or stressful conditions, physiological conditions may also promote development of an cellular stress response.117 For instance, exercise training reduces the extension of lipid peroxidation during acute exercise which has been attributed to induction of oxidative stress.118, 119 Likewise, an enhanced repairing capacity was observed in lymphocytes of workers which were occupationally become exposed with low levels of ionizing radiation.120

It is now clear that sub-lethal doses of oxidants are capable of inducing cellular stress responses in cells. This phenomenon was initially discovered in bacteria, but now it has also been documented in eukaryotic cells.121-124 The protective responses induced in cells during challenge with sub-lethal doses of oxidants have been identified in three major systems. These include hydrogen peroxide (H2O2) and superoxide anion (O2−)-induced reactions in bacteria, protective responses induced by sub-lethal doses of oxidants in eukaryotic cells which render them resistant to lethal doses of the same or a related oxidant and finally protective responses induced by sub-lethal doses of oxidants in eukaryotic cells which render them resistant to lethal doses of other toxic agents. Overall, cells possess two primary defence mechanisms against oxidative stress. The first includes cellular molecules or enzymes that directly participate in scavenging free radicals and preventing oxidative stress-induced damage to cells such as catalase, superoxide dismutases (SOD), glutathione peroxidases, ascorbate and glutathione. The second line, however, consists of enzymes involved in repairing or scavenging oxidatively damaged macromolecules such as DNA and proteins. Typical examples of such enzymes are DNA nucleases and glycosylases.117

4 MECHANISMS UNDERLYING ELF-EMF-MEDIATED CELLULAR STRESS RESPONSE

The cellular stress response to oxidative stress in mammalian cells consists of seven main pathways including unfolded protein response (UPR), antioxidant response, heat-shock response, autophagic response, NF-kB inflammatory response, sirtuin response and DNA repair response. Numerous studies in literature have reported that exposure to ELF-EMF can activate most of these pathways without inducing significant increase in cell death or apoptosis both in normal and in cancer cells (Table 1). Here, we will comprehensively review the ways through which cells respond to elevated ROS following exposure to ELF-EMF and orchestrate cellular stress response (Figures 1 and 2).

TABLE 1. Different cellular stress responses affected by ELF-EMF exposure Experiment performed ELF-EMF treatment Cell line Observed effects 1. Heat-shock protein response Corallo et al.140 100 Hz Primary osteoarthritic chondrocytes Increased Mn-superoxide-dismutase and heat-shock proteins expression Alfieri et al.141 50 Hz, 0.68 mT Endothelial cells A poor and transient activation of HSF1 Frahm et al.142 50 Hz, 1 mT Mouse macrophages Hsp70 and Hsp110 exhibited increased levels at certain time point Wei et al.143 15 Hz, 2 mT Hypoxic cardiomyocytes Significantly increased HSP70 mRNA expression Bernardini144 50 Hz Porcine aortic endothelial cells

Increase in the mRNA levels of HSP70

No increase in Hsp27, Hsp70 and Hsp90 protein levels

Akan et al.145 50 Hz, 1 mT THP-1 cells Increased hsp70 levels in a time-dependent manner 2. Unfold protein response Chen et al.161 Picosecond pulsed electric fields HeLa cells Affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of GRP78, GRP94 and CHOP Keczan et al.162 PEMF HEK263T No remarkable effect HepG2 No remarkable effect HeLa Increased BiP, Grp94 and CHOP expression 3. Autophagy Chen et al.173 Pulsed electromagnetic fields (2 mT, 50 Hz) Embryonic fibroblasts (MEF) A significant increase in autophagic biomarkers including LC3-II and formation of GFP-LC3 puncta was observed 4. NF-kB activation Kim et al.204 RAW264.7 cells Enhanced translocation of phosphorylated NF-κB in to the nucleus and induction of inflammatory responses 5. SIRT3 activation Falone et al.196

ELF-EMF

1 mT, 50 Hz

SH-SY5Y Upregulation of the major sirtuins, increased signalling activity of the NRF2 image

ROS-mediated apoptosis signalling pathways: (1) Accumulation of ROS affects p53 protein which in turn inhibits Bcl-2 and Bcl-XL proteins function and promotes the activity of Bad, Bax, Bak, Puma and Noxa proteins. (2) ROS can induce phosphorylation of JNK. Phosphorylated JNK can activate transcription factors such as SMAD3 and ATF2. Phosphorylated JNK can also translocate to the nucleus and activate C-Jun phosphorylation which in turn can activate transcription of several pro-apoptotic factors. (3) Accumulation of ROS inhibits PI3K-mediated activation of AKT. (4) Accumulated ROS promotes ER stress and expression of CHOP through activation of ATF-4 which in turn can promote Bax activity and inhibit Bcl-2. (5) All these pathways end in the release of cytochrome c which in turn can activate caspase 9 and caspase 3 and result in cleavage of PARP and induction of apoptosis

image

ROS-mediated cellular stress response: (1) mild accumulation of ROS inhibits NADPH oxidase activity. (2) Mild accumulation of ROS activates antioxidant defence system which involves activation of transcription factors including NF-kB, Nrf-1 and AP-1 which in turn upregulates expression of thioredoxin reductase, glutathione peroxidase, SOD, etc., which can suppress further accumulation of ROS. (3) Mild accumulation of ROS activates ER stress through affecting IRE6, ATF6 and PERK. PERK in turn inhibits general protein synthesis and ATF4 and functional XBP-1 promote chaperon transcription, UPR genes and ERAD genes which can protect cells against accumulated ROS. (4) Mild accumulation of ROS can directly induce autophagy through inhibition of mTORC. (5) Mild accumulation of ROS can upregulate expression of HSP70 which can affect protein folding, proteasome activation and induction of autophagy. (6) Mild accumulation of ROS can also activate JNK and after that c-JUN which can in turn activate BECN1, Atg4 and MAP1LC3B genes expressions, most important proteins involved in autophagy. ROS can also inhibit PI3K pathway and modulate autophagy. Finally, mild accumulation of ROS can induce specific decrease in RNA stability and result in mitochondrial activity shut down

4.1 Heat-shock response

In most eukaryotes, heat-shock factors (HSF) [ie transcription factors that regulate expression of heat-shock proteins (HSPs)] are located in cytoplasm in bond with HSP70, HSP90 or other proteins which renders them to be inactive during normal condition.125, 126 During stressful condition however, cells are exposed to a much higher extent of denatured proteins. In this condition, as HSPs prefer to act more like a molecular chaperone instead of a regulatory protein, they become detached from HSF and undergo oligomerization. In next step, oligomerized HSFs translocate to the nucleus where they promote expression of HSP and related heat-responsive genes.127, 128 Different studies have shown that treatment of cells with H2O2 and induced ROS can increase expression of heat-responsive genes.129-131 In the study performed by Volkov et al.128 for example, it was shown that heat treatment and H2O2 elevate expression of AtHSP17.6 and AtHSP18.6 genes up to a similar level. In this regard, it has been hypothesized that heat may also activate HSFs by elevation of ROS in an indirect manner. Consistent with this finding, it has been shown that sub-lethal amounts of ROS induced by thermal stress can enhance segregation of HSP-HSF complexes.126 In addition, certain HSFs have shown to play as a sensor for H2O2.132, 133

Among different ROS, H2O2 is the main player in modulating signalling pathways partly owing to its moderate reactivity and consequently long half-life and stability.134 Furthermore, produced H2O2 can also easily pass through membrane and therefore take role of a sign

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