I. Introduction
Considering the large differences in volume, water content, and other specifications between cancer cells and normal cells, the cell membrane of the former exhibits certain unique charging characteristics, i.e., electromagnetic sensitivity specificity [1, 2].
In this context, it should be noted that changes in external electromagnetic fields may not only lead to changes in the trajectory of charged particles in an organism but also affect the cell membrane potential and organelle membrane potential, among other factors. Such changes can affect the functions of membrane proteins, leading to changes in cell membrane permeability and in specific ion channels in the cell membrane, ultimately affecting the regulation of cellular functions. Therefore, when pulsed electromagnetic fields (PMFs) act on tumor cells or tissues, they may have different kinds of effects—the positive effect of inhibiting tumor cell proliferation, the negative effect of promoting tumor cell growth, or no specific effect on tumor cell growth [3–5]. Numerous studies have shown that PMFs can inhibit the formation of micro vasculature in tumor tissues, cause edema in mitochondria and in the rough endoplasmic reticulum of tumor cells, improve the anti-cancer ability of human immune cells, as well as change the transmembrane potential of cell membranes and the concentration of calcium ions in membranes [6–8]. However, the mechanism by which PMF irradiation acts on cancer cells is not clear at present, and the changing patterns of the biological effect of cancer cells under the effect of different forms of PMF irradiation is difficult to predict, thus complicating the application of PMFs in cancer therapy.
Studies on the mechanism underlying the inhibitory effects of PMFs on cancer have found that they can be primarily classified into the following: inhibiting cell proliferation, directly killing or inducing apoptosis, altering the cell cycle, and affecting cellular pathways. In terms of inhibiting tumor cell or tissue proliferation, research has shown that the metabolism, mitosis, and high-speed anomalous growth of tumor cells in the irradiated group were all inhibited using PMFs [9]. de Seze et al. [10] demonstrated that unipolar square wave PMFs at 0.18 T and 0.8 Hz reduced the proliferation of isolated cervical cancer cells by 15%. Meanwhile, Alcantara et al. [11] identified a significant effect of treatment with PMF at resonant frequencies on MCF-7 cells for the following genes with regard to specific durations of exposure—RICTOR for 10 minutes, PPARG for 10 minutes, NBN for 15 minutes, and CHEK2 for 5 minutes. Kranjc et al. [12] found that electrochemotherapy using a PMF can induce a 2.3–3.0 day tumor growth delay. Furthermore, Williams et al. [13] observed that 10 minutes of daily 10, 15, or 20 mT PMF irradiation (120 pulses/s) significantly slowed down tumor growth and reduced vascularization compared to mice not exposed to magnetic fields.
Meanwhile, studies conducted on apoptosis induction show that PMF treatment can induce apoptosis in association with an increased number of TUNEL-positive cells [14]. PMFs also have immunomodulatory effects. Since irradiation leads to increased levels of tumor necrosis factor alpha, inducing an antitumor response leads to the activation of the pro-apoptotic pathway induced by the interaction between caspase-8 and Fas [15]. In this context, Yao et al. [16] found that PMFs with 0.1–0.3 T have a significant inhibitory effect on isolated murine myeloma cells, which could lead to extensive cell death, with the degree of cancer cells killed by magnetic fields linked to parameters such as magnetic field amplitude B and magnetic field change rate dB/dt. Meanwhile, Crocetti et al. [17] noted that 20 Hz, 3 mT PMF irradiation induced apoptosis in human breast cancer cells but had no effect on normal breast cells. Furthermore, Wang et al. [18] found that PEMF treatment at 1 mT, 8 Hz significantly inhibited the proliferation of ovarian cancer cells (SKOV3). In contrast, another study identified that the distance and number of SKOV3 migration increased under the effect of 1 mT, 8 Hz or 32 Hz magnetic field, i.e., the effect of the magnetic field at this parameter promoted metastasis and the proliferation of cancer cells [18]. This confirms that the response of organisms to PMFs is closely related to the parameters of the acting magnetic field. In this context, Lee et al. [19] studied altering cell cycle, protein expression, and signal transduction pathways to find that very low-frequency electromagnetic fields cause a delay in the cell cycle. The effect of very low-frequency electromagnetic fields on protein oxidation and 20S proteasome function was investigated by Eleuteri et al. [20], who exposed colon adenocarcinoma (Caco 2) cells to a 1 mT, 50 Hz electromagnetic field environment for 24–72 hours to find that irradiation by PMFs induced a global activation of the catalytic component of the 20S proteasome. The phenomenon was more pronounced 72 hours after magnetic field irradiation, with PMF irradiation inducing an increase in cell growth and protein oxidation, the extent of which correlated with the irradiation time [18]. Many other studies [21–23] have also shown that PMFs affect signal transduction pathways in cancer cells and alter ion binding and transport.
With regard to in vitro tumor induction studies, exposing murine 16/C mammary adenocarcinoma tumor fragments to 20 mT, 120 Hz semi-sine wave pulse signals of variable intensity for 10 minutes was found to reduce the vascular volume fraction and increase the necrotic volume of the tumor [24, 25]. Furthermore, epidemiological studies have shown that exposure to industrial frequency electromagnetic fields significantly increases the prevalence of childhood leukemia [26]. Taking 5 mT PMFs as an example, Akbarnejad et al. [27] identified that 5 mT and 10 mT magnetic fields can inhibit the proliferation of U87 cells, while Loja found that 5 mT PMFs can promote the proliferation of COLO-320DM (colorectal adenocarcinoma) and ZR-75-1 (ductal carcinoma) [28]. Notably, these discrepant findings are most likely the result of an inaccurate description of the electromagnetic field, which features different frequencies and waveforms at the same intensity. Therefore, although it might appear that the irradiation source in these studies was the same, a completely different irradiated magnetic field might have been adopted in reality.
Existing studies on the mechanism of PMFs in cancer cells have mostly focused on the physiological response of cancer cells to specific electromagnetic field amplitude parameters. However, the same PMF amplitude may produce completely different PMFs owing to different magnetic field time scales, frequencies, rise rates, and irradiation energies. Therefore, focusing only on the amplitude of the magnetic field while ignoring the other parameters will compel researchers to relate the same amplitude AC magnetic field, DC magnetic field, and PMFs to the same irradiation source. This would lead to the same magnetic field action producing very different action effects, making it difficult to generalize the research results. This issue has troubled many scholars in terms of clarifying the process of changes in the electromagnetic environment during irradiated cell experiments, further leading to difficulty in generalizing the action mechanism of PMFs for cancer cells.
In this paper, irradiation environments were designed for nanosecond pulsed electromagnetic fields (nsPMFs) and microsecond pulsed electromagnetic fields (μsPMFs) at different frequency bands for rat glioma cells (C6). Based on the constructed PMF environment, the cells were continuously irradiated for multiple days to investigate the relationships among the different magnetic field frequencies, the magnetic field rise rate dB/dt, the magnetic field intensity B, the magnetic field action integral ∑BΔt, and the magnetic field energy action integral ∑B2Δt. Ultimately, the most effective magnetic field parameter action interval for inhibiting cancer cell viability is identified, and a mechanism that describes the actions of PMFs on cancer cells is proposed.
II Materials and Methods
1. Cell Culture
A digested rat glioma C6 cell line (provided by the Department of Biology, School of Life Engineering, Xi’an Jiaotong University) was added to a 96-well plate and cultured at 3,000 cells/100 μL per well. The medium was a high-glucose medium (Sijiqing Company, Hangzhou, China) containing 10% FBS (PAA Laboratories GmbH, Linz, Austria) mixed with 1% antibiotic (penicillin and streptomycin) solution (Life Technologies, Carlsbad, CA, USA). This mixture was incubated in a cell incubator at 37°C with 5% CO2. Exposure experiments were performed after 12 hours of adherence.
2. Cell Morphology Observation and MTT Assay
The cell morphology and cell number of the C6 cells before and after the experiment were observed under an inverted fluorescence microscope.
Cell viability was detected using MTT assay (a yellow dye with a molecular formula of C18H16BrN5S and a molecular weight of 414.32). The 10 μL of MTT dye solution at a concentration of 5 mg/mL was added to each well [9], which was then incubated at 37°C in a 5% CO2 incubator for 4 hours. Next, 100 μL of dimethyl sulfoxide was added to each well to dissolve the cell membrane and the formazan in the cells. The absorbance value (optical density [OD]) was measured at a wavelength of 570 nm using a microplate reader. Each dataset was normalized to 100% of the OD value of the control group.
3. Experiment 1: nsPMF Irradiation Experiment
3.1. nsPMF irradiation experiment device
Since the rise time and duration of nsPMFs is extremely short, a transmission-line type square wave electromagnetic field generation device was employed for irradiation. As a result, the magnetic field generation method used a straight wire configuration. For the irradiation experiments, the cells were wall cultured in 96-well plates. The experimental equipment are shown in Fig. 1(a). The test current was 5 A. The magnetic field generator and the magnetic field intensity in the 96-well plate are shown in Fig. 1. The y- and z-coordinates represent the spatial coordinates of the magnetic field measurement point, with the magnetic induction intensity decreasing with an increase in the horizontal distance from the transmission line. When the horizontal distance is fixed, the same magnetic field intensity can be directed at each column of holes, ensuring that the cells in each column of holes are irradiated at the same magnetic field intensity. Furthermore, a DC high-voltage output signal charged the coaxial cable through a coaxial switching device. After charging was complete, the output of the coaxial cable was used as the input of the coaxial switching device, while the output was directed to the ground through a discharge switch or matching resistor to form a square wave current. To realize the steeply rising edge of the nanosecond signal, a mercury switch was employed as the discharge switch. It was installed in the coaxial device to ensure that the output signal was not distorted.
Notably, the electromagnetic fields considered in this article were all generated by current induction. The nanosecond electromagnetic field was generated by a square wave power supply with repetition frequencies of 1 Hz, 5 Hz, 10 Hz, and 20 Hz. Due to the nanosecond waveform being induced by a long straight wire, the magnetic field intensities of the eight holes in each column were the same. As the distance increased, the magnetic field strength declined. Considering the same discharge power source, four control groups with different magnetic field intensities were simultaneously generated at a repetition rate of 6 for each group (the bottom and top samples were discarded due to the edge effect). The nanosecond experiment involved 3 repetitions per group, with the number of identical conditions being 6. Therefore, n was set to 18 (3 × 6) for the nanosecond experiment.
For the irradiation experiments, the cells were first wall-cultured in 96-well plates at a certain concentration for 24 hours, after which the plates were placed in an irradiated magnetic field, as shown in Fig. 1(b). Since the cells in the wells, placed at equal horizontal distances from the long straight wire, were irradiated by the same magnetic field, the magnetic field irradiation intensity in every column of the 96-well plate was the same. The magnetic field amplitude, duration of action, and frequency were selected as the independent parameters to examine the response characteristics of cancer cells to the actions of electromagnetic fields with different parameters. Since the nsPMF irradiation experiment was conducted in a cell room at room temperature (23°C), both the experimental control group and the room temperature control group were set up to exclude the effect of room temperature on cells.
3.2. nsPMF irradiation experimental conditions
Experiments on both the experimental and control groups were conducted at room temperature. Furthermore, another incubator control group was set up. The magnetic induction intensity values for the different columns of cells in the 96-well plate are shown in Table 1. The duration of the magnetic field irradiation was 2 hours. Cell morphology was observed before and after the irradiation experiment using light microscopy, and cell viability tests were performed on the cells after the irradiation experiment. The microsecond experiment involved 5 repetitions per group, with the number of identical conditions being 4 and n being 20 (5 × 4).
The nsPMF waveform in the irradiation experiment is shown in Fig. 2. The magnetic field waveform formed in the first column of the 96-well plate from the coaxial cable, achieving a rise time of 10 ns and a duration of 600 ns. Based on the magnetic field waveforms, the values of each magnetic field parameter—dB/dt, B, ∑BΔt, and ∑B2Δt—were calculated for the different magnetic field effects, as shown in Table 2. The irradiated magnetic field rise rate (T/s) range was [2.58 × 107, 8.45 × 107], the magnetic field amplitude (mT) range was [0.240, 0.787], the magnetic field action integral (mT·s) range was [1.92 × 10−7, 6.29 × 10−7], and the magnetic field energy action integral (mT2·s) range was [3.35 × 10−8, 3.60 × 10−7].
4. Experiment 2: μsPMFs Irradiation Experiment
4.1. μsPMFs irradiation device
To ensure a relatively uniform distribution of the magnetic field in the irradiated cell, a solenoid was adopted as the carrier for the magnetic field. During the irradiation experiments, the cells were wall cultured in 96-well plates. The experimental diagram is shown in Fig. 3. A magnetic field environment characterized by different current waveforms and amplitudes was chosen for the experiment. The various parameters of the magnetic field, including amplitude, rise time, and action integral, represent its different properties. Furthermore, during the experiment, the control cells were placed in a separate standard incubator without irradiation. In this context, the microsecond electromagnetic field could be generated using a square wave or an exponential wave current source. A cloud diagram of the solenoid magnetic field simulation calculation is presented in Fig. 3. Notably, the magnetic field strengths of the four holes in the middle were the same. Hence, they were considered the experimental group.
4.2. μsPMFs irradiation experimental conditions
Solenoids featuring different numbers of turns were used in the experiment to simultaneously generate uniform magnetic fields of different intensities when the same current was applied. Notably, the short duration of the microsecond current implies that the loop could carry a small amount of inductance. As a result, the number of turns for the solenoid used in this experiment was set to 70 turns and 20 turns. The cells placed in the solenoid were accompanied by a thick, insulating cylindrical block at the bottom. Furthermore, the cells located in the central region of the experiment were selected to ensure that they were in a relatively uniform magnetic field environment, as shown in Fig. 3(b). Taking 531 A current and the 70-turn solenoid internal magnetic field as an example, it was observed that the magnetic field intensity in the test sample area formed a uniform magnetic field of about 311 mT. The experimental conditions were as follows: Irradiation treatment for 4 hours per day for a total of 4 days. The current amplitude and magnetic field irradiation intensity used in the experiment are listed in Table 3.
The current and magnetic field waveforms of the exponential and square waves selected for the irradiation experiment are shown in Fig. 4. The magnetic field waveform was located 3 cm from the bottom of the solenoid. The rise time of the exponential waveform is 85 μs, and the waveform duration is 205 μs. Meanwhile, the square wave exhibits a rise time of 10 μs and a duration of 100 μs. Moreover, the intensity of the generated pulsed electromagnetic induction was found to be linearly related to the pulse output current amplitude.
Based on the measured μsPMFs waveforms, the values of each magnetic field parameter—magnetic field rise rate dB/dt, magnetic field amplitude B, magnetic field action integral ∑BΔt, and magnetic field energy action integral ∑B2Δt—under the action of μsPMFs exponential and square wave magnetic fields were calculated, as shown in Table 4. The μsPMFs attained a magnetic field rise rate (mT·s) range of [4.493 × 102, 1.329 × 105], a magnetic field amplitude (mT) range of [16.4, 854], a magnetic field action integral (mT·s) range of [2.4 × 10−3, 0.0824] and a magnetic field energy action integral (mT2·s) range of [0.029, 62.545]. Furthermore, the rise rate of the microsecond magnetic field was faster.
The magnetic field measurement system employed in this study consisted of a detection coil and an oscilloscope. Notably, the inner conductor of the coaxial cable was turned into the detection coil, which was then set to have 1 turn and a diameter of 10 mm. The measuring coil was placed into the measured magnetic field according to the law of electromagnetic induction (Eq. 1). The waveform of the induced electromotive force was displayed in the oscilloscope.
where ɛ is the induced electromotive force, N denotes the number of coil turns, φ refers to the magnetic flux, B is the magnetic intensity, and S signifies the cross-sectional area of the detection coil.
According to Eq. (2), the waveform of a magnetic field can be obtained by integrating the induced electromotive force. In this study, the measured value of the magnetic field deviated from the calculated value by less than 3%.
where ɛ is the induced electromotive force, N denotes the number of coil turns, B is the magnetic intensity, and S signifies the cross-sectional area of the detection coil.
5. Statistical Analysis Methods
The t-test and the honestly significant difference (HSD) test were the two statistical tests conducted in this paper. The t-test (Student t-test) was mainly conducted on the normally distributed samples with small sample content (e.g., n < 30) and an unknown standard deviation σ of the total. The purpose was to compare the unknown total mean μ, represented by the sample mean, and the known overall mean μ. The corresponding formula is defined in Eq. (3):
where t denotes the test statistic, n refers to the number of samples, X̄ is the sample mean, μ0 signifies the overall mean, and S is the sample standard deviation.
Meanwhile, the HSD test compares the mean of all pairs simultaneously to calculate a single value that lends itself to the comparison of the difference between the mean values of all pairs. This value is called the HSD, and it can be expressed using the following equation:
where a denotes the significance level, k signifies the number of means in the experiment, and n–k is the error degree of freedom. Notably, any difference between the means that exceeds the HSD value indicates that the difference between the means is significant.
III. Results
1. Results and Analysis of the Effects of nsPMFs on Cancer Cells
The cell morphologies of the irradiated and control groups were observed under a light microscope, as shown in Fig. 5. The cells in the irradiated group with different magnetic field intensities and frequencies were selected for observation in comparison to those in the control group. The comparison revealed no significant difference in the cell morphologies of the C6 cells before and after receiving magnetic field irradiation. The cell morphology and growth status were normal, and no cell fragmentation or micronuclei were observed. Furthermore, a comparison of the experimental results for the room temperature control group and the experimental control group showed no significant difference between C6 cells, indicating that low room temperature did not cause significant damage to the C6 cells and had no significant effect on the results of the irradiation experiments.
After two hours of irradiation, cell viability assays were performed, the results of which are shown in Fig. 6. The C6 cells exhibited a window effect with regard to both nsPMF intensity and frequency. At a magnetic field frequency of 1 Hz, as shown in Fig. 6, the magnetic field effect at all intensities exhibits an inhibitory effect on C6 cells. With a change in the magnetic field frequency, the magnetic field at 0.240 mT continues to show an inhibitory effect on the cells. Even when the magnetic induction intensity is 0.353 mT, the nanosecond magnetic field exhibits an inhibitory effect on C6 cell viability, except at the magnetic field frequency of 10 Hz. Furthermore, when the magnetic field intensity is greater than 0.353 mT and the magnetic field frequency is greater than 5 Hz, no significant difference between the cell viability of the control and irradiated groups is observed.
The trend of variation in C6 cell viability with regard to the magnetic field parameters under the effect of nsPMFs shows that the magnetic field inhibited the viability of C6 cells better under the effects of low field intensity and low-frequency magnetic field. At specific intervals of magnetic induction intensity or frequency, the physiological state of the cancer cells changes in response to irradiation by the magnetic field. This phenomenon is defined as the effective window of action of the magnetic field. The difference between the cell viability of each irradiated group and the control group is shown in Fig. 7. Furthermore, the trends of cell viability in response to magnetic induction intensity and magnetic field frequency are shown in Fig. 7. The experimental results show that the magnetic field parameters that achieved the best effect of nsPMFs on cell viability inhibition were 1 Hz, 0.240 mT, with the cell viability being 84.97% for the control group (p < 0.001).
2. Results and Analysis of the Effect of μsPMFs on Cancer Cells
The cell morphologies of the irradiated and control groups were observed under a light microscope before the experiment (Day 0), after the first irradiation session (Day 1), and four days after irradiation (Day 4), as shown in Fig. 8.
The C6 cells exhibited no significant difference in their cell morphologies before and after μsPMFs irradiation. Both cell morphology and growth status were normal. Notably, the growth morphology of the cells was mostly long shuttle-shaped, with a large oval-shaped nucleus located at the center. The cytosol was elongated along two sides. A small number of cells were star-shaped, with round or oval nuclei located at the center, along with many protrusions in the cytoplasm. No fragmentation or micronuclei were observed. Overall, no significant difference between the experimental and control group cells was observed.
Cell viability tests were performed on the control and irradiated cells after 4 hours of irradiation each day, the results of which are shown in Fig. 9. The cells manifested a cumulative effect after being irradiated by electromagnetic fields, with an inhibitory effect appearing after many days of magnetic field irradiation. On the first day, after only 151 mT magnetic field irradiation, C6 cell viability decreased by 8.7% relative to the control group (p < 0.05), which was a significantly different result from the cell viability observed under the effect of other magnetic field intensities. However, the rest of the groups did not exhibit any significant differences from the control group. On the second day, the cell viability values in each group were not significantly different from those of the control group. On the third day, C6 cell viability was reduced significantly by 22.45% for the 311 mT group compared to the control group (p < 0.001), while the rest of the groups showed no significant difference. On the fourth day, the decrease in cell viability was 21.91% (p < 0.01) in the 16.4 mT group, 13.49% (p < 0.01) in the 151 mT group, and 14.7% (p < 0.05) in the 311 mT magnetic field. The results of the Turkey test confirmed the window effect of the amplitude of magnetic field irradiation on C6 cells irradiated by magnetic fields.
Fig. 10 shows the results of testing C6 cell viability and control group cell viability under irradiation by square wave μsPMFs. On Day 1 after the square wave MMF irradiation, cell viability decreased by 11.6% compared to the control group for the magnetic intensity of 45.4 mT. On Day 2, except for the 363 mT group, the cell viability values of all other groups reduced significantly compared to that of the control group. In particular, for the 45.4 mT MMF group, cell viability decreased by 11.32%. Furthermore, under 122 mT μsPMFs and 854 mT MMF, cell viability decreased by 11.45% and 10.89%, respectively. On Day 3, the viability of the C6 cells in the 45.4 mT and 122 mT groups decreased significantly by 18.26% (p < 0.01) and 8.1% (P < 0.01), although there was no significant difference between the other groups and the control group. On Day 4, viability in the 45.4 mT group decreased by 10.79% (p < 0.01), the 122 mT group decreased by 32.53% (p < 0.01), and the 363 mT group decreased by 29.2% (p < 0.05). Therefore, under the effect of square wave μsPMFs, the cumulative effect on C6 cells in response to external electromagnetic stimuli was obvious. Moreover, the cell viability test results for the fourth day indicated that different intensities of the applied magnetic field led to significantly different cell viability of C6 cells in each irradiation group, implying the presence of a significant window effect.
The results of cell viability tests for the C6 cells and for the control group under square wave μsPMFs irradiation are shown in Fig. 11. Fig. 11(a)–11(d) depict the cell viability variation curves with regard to the magnetic field rise rate (dB/dt), magnetic field intensity (B), magnetic field action integral (Bt), and magnetic field energy action integral (B2t), respectively.
Fig. 11 points to a significant window effect and time cumulative effect between cell viability and each magnetic field parameter. The cell viability response to each parameter of the magnetic field showed a significant nonlinear relationship. Notably, a more stable inhibition was identified in the cell viability tests after the third day of the experiment. This phenomenon may be attributed to cell processes, in which the cells first produce a stress response after receiving external electromagnetic irradiation. During this process, the cell regulates the functions of its organelles, such as mitochondria, to adapt to changes in the external environment. When the duration of continuous irradiation grows, the damage to the cells accumulates up to a certain level, after which an inhibitory effect on the cells is registered. The magnetic field parameter ranges for the effective inhibition of cell viability were found to be as follows: a magnetic field rise rate dB/dt (T/s) of [4.14 × 103, 5.65 × 104], a magnetic field action integral Bt (mT·s) of [0.012, 0.045], and a magnetic field energy action integral B2t (mT2·s) of [1.28, 11.3].
IV. Discussion
The experimental results presented above were combined with the results of the millisecond pulsed electromagnetic field (msPMF) irradiation tests performed by Wertheimer and Leeper [26]. It was found that although the amplitudes of nsPMFs are considerably lower than those of μsPMFs and msPMFs, cell viability can be inhibited by just 2 hours of continuous irradiation experiments using nsPMFs.
Furthermore, when comparing the experimental results for nsPMFs with those acquired at different frequency bands, it was evident that although the magnetic field amplitude of nsPMF is much lower than μsPMFs and msPMFs, it has an extremely fast rise time. The different values pertaining to the increasing speed of magnetic field intensity are arranged in high-to-low order in Table 6 [20].
It is observed that there is a negative correlation between the overall trend of cell viability values and the change in magnetic field rise rates. Although the magnetic field rise rates in groups (e) and (g) are higher, their cell viability is also higher than that of groups (d) and (f), respectively. This result can be attributed to the fact that groups (e) and (g) belong to the nsPMF experimental group, whose magnetic field action integral values are smaller than those of the other experimental groups, although their magnetic field has a higher rise rate. For the experimental groups (d), (e), (f), and (g), the rate of rise of the magnetic fields is similar to each other in the order of 105. At this point, the intensity of PMFs in the experimental groups with larger action integral values correspond to lower cell viability. For instance, in the case of the msPMF experiments (groups (a) and (b) in Table 6), while the values of Bt and B2t were higher, the inhibitory effect on cells was the weakest for this group compared to the nanosecond and microsecond magnetic field irradiation experiments due to their lower rate of magnetic field rise. This indicates that the rise rate (high-frequency component) of PMFs plays a major role in terms of their effect on cancer cells at different scales.
Studies conducted by other researchers have also shown that when PMFs are applied to cells, the cell membrane becomes equivalent to a low-pass filter due to the presence of cell membrane capacitance [30, 31]. Drawing on the analysis of the experimental results obtained in this paper, it can be established that the low-frequency component of a magnetic field primarily acts on the cell membrane, while having basically no effect on the nucleus, mitochondria, and other organelles inside the cell membrane. However, as the frequency increases, PMFs penetrate the cell membrane to enter the interior, thus affecting the nucleus. When the frequency is very high, the high-frequency component can pass through the nuclear membrane and have a strong destructive effect on the nucleolus.
The results of the experimental groups in response to different Bt values are presented in high-to-low order in Table 6. The order of magnitude variation of Bt in the ranges from 10−7 to 101 mT·s. Notably, cell viability did not show a significant correlation with an increase in the integral value of the magnetic field effect. Similarly, the relationship between cell viability and the magnetic energy action integral B2t (mT2·s) remained the same. The above data indicate that the energy accumulation of magnetic fields is not the main cause for PMF action on cancer cells. The effects of PMFs on the cells employed in this paper were all non-thermal, and the accumulation of energy was not sufficient to produce a strong damaging effect on the cells. In terms of the overall effect, a stronger action integral and energy action integral did not lead to better inhibition.
Therefore, the most important parameter involved in the action of PMFs on cancer cells is the rate of rise of the magnetic field. This finding may be explained by the fact that PMFs with a faster rise rate generate a stronger induced electric field inside the cell, which can change the cell membrane potential, as well as the distribution of the organelle membrane potential, leading to a series of changes in the physiological activity of the cell.
V. Conclusion
In this paper, nanosecond and microsecond pulsed electromagnetic action devices were designed by combining square and exponential waveforms to produce a pulsed electromagnetic environment characterized by different magnetic field rise rates, intensities, action durations, and energy accumulation. Based on this environment, the responses of C6 cells to different magnetic field parameters under the effect of different scales of PMFs were estimated, and the effective window values corresponding to each magnetic field parameter were obtained. When nsPMFs were applied, the low-frequency and low-intensity electromagnetic fields were found to be more likely to inhibit the proliferation of C6 cells. Furthermore, when the magnetic induction intensity was increased to more than 0.353 mT and the magnetic field frequency was greater than 5 Hz, no significant change in cell physiological activity was observed.
This study identified a significant window effect and a time cumulative effect between cell viability and each magnetic field parameter when electromagnetic fields were applied at the microsecond scale. The magnetic parameters dB/dt (T/s), Bt (mT·s), and B2t (mT2·s) for the effective inhibition of C6 cell viability were [4.14 × 103, 5.65 × 104], [1.18 × 10−2, 4.51 × 10−2], and [1.28, 11.30], respectively. The experimental results of the msPMFs were also combined to those of the nsPMFs and μsPMFs to analyze the mechanism of the action of PMFs on C6 cells. The results showed that the change in PMF rise rates (dB/dt) significantly influences the effect of magnetic fields in inhibiting cancer cell proliferation. Furthermore, when the difference of dB/dt is small, the magnetic field action integral becomes the main factor affecting cancer cell viability.
The effect of the pulsed magnetic field on cancer cells, considering different parameters, can be employed to establish a basis for the subsequent design of the pulsed magnetic field. Furthermore, we may be able to arrive at better ideas for targeting and regulating the physiological state of cancer cells by conducting electromagnetic modeling analysis for cancer cells. In this context, the combined action of PMFs and plasma to enhance the killing effect may also be considered a further area of study. Overall, the results of this study offer an experimental basis for the selection of magnetic field parameters for PMF treatment of cancer cells and establish a theoretical basis for the electromagnetic therapy of cancer.
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