Mitochondrial ROS Release and Subsequent Akt Activation Potentially Mediated the Anti-Apoptotic Effect of a 50-Hz Magnetic Field on FL Cells

Extremely-low frequency electromagnetic fields (ELF-EMF) generally refer to EMF with frequency ranges from 0-300 Hz. With the rapid increase in usage of electricity in the last several decades, ELF-EMF, particularly 50/60 Hz power frequency fields, exists profusely in the public and occupational environments, and there has been substantial concern about the possible detrimental health effects of ELF-EMF. Some epidemiological studies have demonstrated correlations between exposure to ELF magnetic field (MF) and increased risks of developing cancer and other disorders [1,2,3]. On the basis of these findings, the International Agency for Research on Cancer (IARC) has classified ELF-MF as a ‘possible human carcinogenic agent' (class 2B). However, the mechanisms are still unclear.

It is well known that apoptosis disorder of cells is generally related closely to tumor genesis. Circumventions to apoptotic signaling may confer on cells an unwarranted survival and proliferative advantage and contribute to the transformation of normal to cancerous tissue [4]. Several studies have demonstrated that MF exposure could directly enhance cell proliferation [5,6]. In addition, MF exposure was also reported to have an anti-apoptotic effect when combined with other apoptosis inducer [7]. It has been hypothesized that MF might affect biological systems by increasing cellular free radical activity which in turn causes transition of membrane structures and permeability to small molecules or interfering with certain signaling pathways [8].

Recently, we found that exposing Human amniotic (FL) cells to a 50-Hz MF induced a moderate increase in reactive oxygen species (ROS) that in turn caused a transient mitochondrial permeability transition (MPT) by opening mitochondrial permeability transition pores (mPTP) [9]. It is believed that a transient mode of MPT could prevent accumulation of ROS inside mitochondria and consequential damage of that organelle [10]. However, the biological implication of MF-induced transient MPT is poorly understood. In this study, effects and possible mechanisms of transient MPT induced by a 50-Hz MF on cell early apoptosis and cell viability, and combined effects of MF exposure followed by staurosporine incubation were investigated using the mitochondrial ROS-specific dye MitoSOX and a MPT inhibitor.

The main reagents and antibodies used in this experiment were: Staurosporine (STS) (Gene, HongKong, China), Bongkrekic acid (BKA) (Enzo Life Sciences, Farmingdale, NY, USA), N-acetyl-L-cysteine (NAC) (Beyotime Institute of Biotechnology, Shanghai, China), LY294002 (Beyotime Institute of Biotechnology, Shanghai, China), SB216763 (Sigma-Aldrich, St. Louis, MO, USA), Cyclosporine A (CsA) (Yuanye Bio-Technology, Shanghai, China), U0126 (Cell Signaling Technology, Beverly, MA, USA), anti-phosphorylated Akt (Ser⁴⁷³) antibody (Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH antibody (Cell Signaling Technology, Beverly, MA, USA), goat anti-rabbit antibody (LI-COR, Lincoln, Nebraska, USA).

Human amniotic (FL) cell line (kindly provided by the Department of Pathophysiology, Zhejiang University School of Medicine) was cultured in Minimum Essential Medium (Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (Sijiqing Biotech, Hangzhou, Zhejiang, China) at 37 °C in a humidified atmosphere with 5% CO₂. Cells were seeded 24 h prior to an experiment. For flow cytometry and Western blot studies, cells were seeded on 60-mm petri dishes (Corning, NY, USA) at a density of 2×10⁵ and 8×10⁵ cells/dish, respectively. For mitochondrial ROS assessment and cell viability analysis, cells were seeded at a density of 1.5×10⁴ cells/well in 96-well plates (Greiner Bio-One, Frickenhausen, Germany).

The exposure system (sXc-ELF) (Fig. 1A and B) used in the present experiment was designed by the Foundation for Information Technologies in Society (IT'IS, Zurich, Switzerland) [11]. Briefly, it consisted of two exposure chambers which were put in a CO₂ incubator, and a set of control device outside the incubator. Each chamber was composed of contained a set of square Helmholtz coils (20×20 cm²) which were double-wrapped with two lines of copper wire, and was encased by mu-metal which shielded the cells in it from stray MF. A fan on the metal wall ventilated and made air and temperature uniformity between chamber and incubator. Current was fed into the coils with the same direction in the exposure chamber whereas opposite direction currents were fed into the coils in the sham exposure chamber. The magnitude and direction of current which was fed into each set of Helmholtz coils was set by the computer, with one set for ‘MF exposure' and the other ‘sham exposure'. The conditions of the setting (exposure of sham) were blind to the experimenters who did the assays. When feeding a line current to the coils, a 50-Hz sinusoidal magnetic field was generated in the center of the coils in the exposure chamber, yet there was almost no 50-Hz MF at the center of the sham exposure coils. In both chambers, the total static MF was 18.5 μT with a 14.1 μT horizontal and 12.0 μT vertical components. The flux densities were measured using an EFA-300 EM Field Analyzer (Narda Safety Test Solutions, Germany). The heterogeneity of MF distribution within the 20×20×20 cm³ space in the exposure chamber is less than 1%. The sham isolation rate is more than 43 dB and E-fields is less than 1 V/m.

During exposure, the incubator was aired with 95% humidity air and 5% CO₂. The temperature in the chambers was recorded real time and kept at 37.0 ± 0.1 °C throughout the entire exposure period. The exposure system was turned on at least 2 h before an experiment. After exposure, dishes were removed from the chambers while the magnetic field was still on. During exposure, cell dishes were put in the center of the coils. The MF was perpendicular to the dishes. In the experiments, cells were exposed to a 50-Hz MF at 0.4 mT (rms) for 5, 15, 30, 60, or 120 min with corresponding parallel sham exposure groups.

Apoptosis was assessed using the Annexin V Apoptosis Detection Kit (Multiscience, Hangzhou, Zhejiang, China) by flow cytometry (Beckman Coulter, CA, USA). After treatment, FL cells were trypsinized, washed with cold PBS, and re-suspended with 500 μl binding buffer containing 5 μl Annexin-V-FITC and 10 μl PI. After incubation for 5 min at 37 °C in the dark, fluorescence levels were quantified by flow cytometry. The number of experimental runs was 5.

Cell viability was assessed by a CCK-8 kit (Dojindo, Kumamoto, Kyushu, Japan) according to the manufacturer's protocol. Briefly, cells were seeded 24 h prior to an experiment at a density of 1.5×10⁴ cells/well in 96 well-plates. After MF/sham exposure, 10 μl reagent of the kit was added into each well. Cells were incubated for 80 min at room temperature. Then, OD value was read using a multimode plate reader (Thermo Scientific, NY, USA) at the wavelength of 450 nm. The number of experimental runs was 4, and in each experimental run, 5 replicate samples were used per experimental condition.

Mitochondrial ROS level in cells was measured by the MitoSOX Red kit (Molecular Probes, Carlsbad, CA, USA) according to the manufacturer's procedures. Briefly, cells seeded in 96-well plates were incubated with MitoSOX Red probe at a final concentration of 5 μM for 10 min at 37 °C in the dark, and then gently washed three times by pre-warmed PBS before MF exposure. After exposure, fluorescence level was quantified by a multimode plate reader (Thermo Scientific, NY, USA) at excitation/emission wavelengths of 510 nm/580 nm. The number of experimental runs was 5, and in each experimental run, 5 replicate samples were used per experimental condition.

After MF/sham exposure, FL cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1 mM PMSF and protease inhibitor cocktail and centrifuged at 12000 g for 10 min at 4 °C. Protein concentrations were determined by BCA protein assay (Beyotime Institute of Biotechnology, Shanghai, China). Equal amount of protein (25 μg/lane) from each sample were separated by 10% SDS-PAGE and transferred to NC membranes (Whatman, Dassel, Germany). After blocking with 5% non-fat milk for 2 h, the membranes were incubated with the primary antibodies overnight at 4 °C and corresponding secondary antibodies for 1 h at room temperature. The blots were quantified with an Odyssey infrared imaging system (LI-COR, Lincoln, Nebraska, USA). The number of experimental runs was 4.

Date analysis was performed using the SPSS 21 software package. Each data point is the averaged data of at least three independent experiments. Statistical comparisons among groups were evaluated by unpaired t-test for two-group comparisons or one-way analysis of variance (one-way ANOVA) followed by SNK test for multiple-group comparisons. A P-value less than 0.05 was considered statistically significant.

MF exposure has been reported to affect cell fate in a bimodal fashion, either cytotoxic or cytoprotective [12,13]. To assess whether MF exposure had any effects on cell death or cell viability, FL cells were exposed to a 50-Hz MF at 0.4 mT for 30, 60, or 120 min, then cell early apoptosis and cell viability were determined by Annexin/PI staining and CCK-8, respectively. The results showed MF exposure had no significant effect on cell early apoptosis or cell viability (Fig. 2). Furthermore, we also determined early apoptosis when FL cells were cultured for different time periods after exposure to 0.4 mT MF for 60 min. As results shown in Fig. 2B, MF exposure still had no significant effect on cell early apoptosis. However, it is noteworthy that the results of CCK-8 kit depending on redox potential of tetrazolium salts might be misleading under the condition of MF exposure which could affect cellular redox state. Thus the potential MF-effects on viability would be questionable. But anyway, the MF alone did not affect the early apoptosis as revealed by Annexin V.

In view of ELF-MF is a kind of weak physical stressor, the possible combined effects of MF with other factors were explored in the present experiments. Results of flow cytometry showed that FL cells treated with staurosporine for 4 h led to a dose-dependent early apoptosis stained by Annexin/PI (Fig. 3A). However, pretreatment with MF exposure for 60 min significantly reduced the early apoptosis induced by a relative low dose of staurosporine (0.1 μM) (Fig. 3B and Fig. 4). The data indicated that MF exposure has an anti-apoptotic role. Additionally, when FL cells were exposed to MF at different magnetic flux intensities from 0.2 to 2 mT for 60 min followed by staurosporine incubation for 4 h, we observed that only MF pre-exposure at 0.4 mT and 1 mT showed the anti-apoptotic effect (Fig. 3C). These data indicated that the anti-apoptotic effect appeared to be non-linear. There were exposure-duration and intensity windows.

Although a burst of mitochondrial ROS production is usually detrimental to cells, studies showed that a moderate increase might contribute to the activation of some protective pathways [14]. In the present study, mitochondrial ROS level was detected using MitoSOX, a specific mitochondria-targeted probe. We found that mitochondrial ROS production increased after exposure to a 0.4 mT MF for 30 and 120 min, but there was no significant change after 60 min of exposure (Fig. 5A). Our previous study had revealed that exposure to a MF at 0.4 mT for 60 min could induce MPT [9], which usually is a main pathway for mitochondrial ROS release into cytoplasm. In order to test the possibility that MF increases mitochondrial ROS releases via MPT, FL cells were exposed to 0.4 mT MF for 60 min with pretreatment of CsA, a specific MPT inhibitor. As the results shown in Fig. 5B, CsA pretreatment effectively increased mitochondrial ROS, i.e. CsA inhibited mitochondrial ROS release into the cytoplasm. Then, the possible roles of mitochondrial ROS in the anti-apoptotic effect of MF exposure were further investigated with MPT inhibitors (BKA, CsA and SB216763) and ROS scavenger (NAC). The results indicated that both MPT inhibitors and ROS scavenger pretreatments significantly attenuated the anti-apoptotic effect of MF exposure on FL cells (Fig. 5C).

The PI3K/Akt pathway is one of the protective pathways which usually are activated by ROS, and Akt has been recognized as a key player in the regulation of cell growth and proliferation as well as in cell metabolism [15]. In the present study, we found that exposing cells to a 50-Hz MF at 0.4 mT for 60 min enhanced Akt phosphorylation at Ser⁴⁷³ (Fig. 6A and B) which could be abolished by pretreatment with either MPT inhibitors BKA and CsA or a ROS scavenger NAC (Fig. 7). This indicated that Akt activation might be a downstream event of mitochondrial ROS release. In addition, pretreatment with the PI3K/Akt signaling inhibitor LY294002 also abolished the anti-apoptotic effect of MF (Fig. 6C), confirming that Akt activation mediated the anti-apoptotic effect of MF exposure on cells.

In our previous research, MF exposure was found to induce transient MPT of which the possible biological effects had not been fully explored [9]. The results of this present study demonstrated that MF exposure alone had no significant effect on cell early apoptosis or viability, but it could attenuate early apoptosis induced by staurosporine with time and intensity windows. The data supports the opinion that biological responses to ELF-EMF might be characterized by ‘window' effects with respect to frequency, amplitude, and exposure duration exposure which were observed before in other biological systems [16,17,18]. The results also revealed that this anti-apoptotic effect of MF was mediated by Akt signaling, and mitochondrial ROS release into the cytoplasm through mPTP was possibly required for Akt activation during MF exposure.

Some studies have reported before that MF could elicit proliferative response in various cell types [19,20]. Since no direct tumorigenic or mutagenic effect has been attributed to MF [21], it has been hypothesized that MF played the role of a tumor promoter in the presence of a primary initiator via different mechanisms, particular that involved in apoptosis [22]. For example, Robison et al. [23] demonstrated that cancer cell lines exposed to MF (60 Hz, 0.18 mT) for 24 h exhibited a decrease in the percentage of cells undergoing apoptosis induced by heat shock, accompanied by a marked decrease in DNA repair rate, validating that MF exposure could not only alter the apoptotic process but also induce genomic instability. But how MF exposure exerts its anti-apoptotic effect is less clear. In the present study, we found that MF exposure enhanced mitochondrial ROS production and protected FL cells from staurosporine-induced apoptosis. It seems that the possible biochemical mechanism responsible for changes in the apoptotic process in exposed cells may be related to a redox mechanism. Interestingly, Nicola et al. [7] showed that exposure to MF at 0.1 mT significantly decreased glutathione intracellular level and protected U937 cells from apoptosis induced by puromycin. This finding is in agreement with the effects observed in our study, i.e. MF exposure elicited anti-apoptotic effects involving redox mechanisms. Since a decrease in glutathione or rise of ROS production could enhance intracellular ROS level respectively, thus, it is imperative to study which of these processes is responsible for the anti-apoptotic effect. We showed for the first time that the anti-apoptotic effect of MF on FL cells was possibly dependent on mitochondrial ROS release into the cytoplasm through transient mPTP opening (namely MPT). This might explain why the anti-apoptotic effect was restricted at certain exposure-duration and intensity, as mitochondrial ROS release was concomitant with mPTP opening which occurred only under certain exposure period or intensity.

MPT is a phenomenon representing a sudden increase in the permeability of the inner mitochondrial membrane allowing not only protons but also other ions and solutes of masses up to 1500 Da to go through the membrane [24]. Depending on the length of duration, MPT plays various roles, bringing cells to different endings [25]. It is noteworthy that in this study, MF-induced mitochondrial ROS release through mPTP transiently and this process itself alone would have little effect on cell early apoptosis or cell viability. For a long time, mitochondrial ROS has mainly been considered for their roles in cell death or senescence. Subsequently, it was showed that within certain limits, mitochondria-derived ROS play diverse and critical roles in the metabolic tolerance to environmental stress through mediating pro-survival signaling pathways [26,27,28]. As expected, we found that suppressing the outflow of mitochondrial ROS effectively eliminated the anti-apoptotic effect of MF. This suggests that induction of MPT and the subsequent release of ROS after MF exposure might serve a survival role. However, one limitation of the present study is that we cannot exclude whether CsA has a non-specific pro-apoptotic effect in FL cells, thus the anti-apoptotic effect of MF was mitigated in the presence of CsA was not convincing enough. The other limitation is involved in the source of ROS. Cytoplasmic ROS originate from other enzyme systems like Nox might still play a role in the mediation of the anti-apoptosis effect of MF. Hence, solid data confirming the origination of ROS are still needed. In spite of this, the present study provided evidence that the MF anti-apoptotic effect was possibly involved in mitochondrial ROS release.

Several studies had demonstrated that MF exposure could activate various signaling pathways, including PI3K, ERK, JNK, and Wnt signaling pathways which related closely with cell fate [29,30,31,32]. The anti-apoptotic effect of MF exposure in the present study was blocked by inhibiting Akt activation, indicating that it was a key step in the protective effect against early apoptosis induced by staurosporine. Furthermore, we showed here that not only the release of mitochondrial ROS was corresponding with Akt phosphorylation, but also that the Akt activation induced by MF exposure could be abolished completely by either a ROS scavenger or MPT inhibitor. This suggests that mitochondrial ROS rising and subsequent release through mPTP might be upstream of and essential for Akt activation in the signaling cascade after MF exposure. So far, the PI3K/Akt signaling pathway has been implicated in the control of cell fate by regulating apoptosis due its ability to inactivate several pro-apoptotic molecules as well as the Forkhead family that induces the expression of other pro-apoptotic factors by affecting their phosphorylation status, and activating the IκB kinase (IKK), a regulator of the survival factor NF-κB [33,34]. In fact, there is a study reporting that MF exposure could promote cell proliferation through regulation of NF-κB activation [8]. However, we do not know, in the condition of the present study, whether Akt exerts its anti-apoptotic effect according to the ‘classic mode', and additional work is needed to explore whether Akt has any cross talking with aforementioned signaling pathways that are activated by MF.

In summary, the current study contributes to the knowledge on the complex effects of MF on cells and provides the first evidence for the potential involvement of transient mitochondrial ROS release and subsequent Akt activation in mediating cell apoptotic events, indicating that the MPT induced by a 50-Hz MF might be critical to cells when synergistically exposed to other apoptotic factors. Under certain conditions, MF is able to reverse apoptotic events.

This study was supported by grants from the Major State Basic Research Development Program of China (973 Program) (No. 2011CB503700) and the National Natural Science Foundation of China (No. 31370831).

The authors report no conflicts of interest.