PART 3. Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanism

PART 3. Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanism                               Discussion

Magnetic feld exposure has the potential to serve as an advanced strategy in cancer management. ROS produced

by cells using this method appear to be the key to tumor growth inhibition³. We proved that the main mechanism

was not only related to ROS but also closely related to the contact between cells. We speculated that the magnetic

feld exposure changed the secretion of certain substrates, leading to the changes in signaling and thus restoring

tumor cell contact inhibition. Based on the experimental results and previous experiments, four properties of

magnetic feld suppression were summarized.

2. The experiment with adherent cells showed that the tumor cells were more susceptible to magnetic inhibi

tion using conditioned media more suitable for cell growth. As shown in Fig. 1b and c, the number of tumor

cells in the conditioned medium of the unexposed control group was significantly higher than in the fresh

medium, while the number of tumor cells in the conditioned medium of the exposed group was significantly

lower than in the fresh medium (the magnetic inhibition effect was significantly higher in the infusion group

than in the change group). It was suggested that the conditioned medium was suitable for not only the

exponential growth of cells in the absence of the magnetic field but also the communication between cells,

which increased the contact inhibition effect under the magnetic field. Hence, the magnetic field inhibition

rate depended on the stability of the environment to extend the contact inhibition.

 

3. We determined that certain substrates in the conditioned media also contributed to magnetic inhibition. The

inhibition was observed when the infusion group (A549) conditioned medium was transferred to unexposed

cells after magnetic field exposure, demonstrating the presence of inhibition signals in the medium. The

suspended tumor cells were less sensitive to magnetic inhibition compared with the adherent tumor cells.

Also, the cell aggregation was inevitably affected during the transfer of the conditioned media. Therefore,

more design and consideration were required for the suspension conditional media transfer of tumor cells.

 

In a xenogeneic cell study in which the conditioned media of normal cells 293 T and tumor cells A549 were

exchanged and transferred to unexposed cells after magnetic field exposure, the tumor cells showed inhibition

while the normal cells did not show significant inhibition. It indicated that certain substrates interacting with

the magnetic field were ubiquitous in cells sensitive to tumor cells. Figure 5c and d indicates that these substrates

also existed in nontumor cells and did not inhibit the growth of nontumor cells. It was speculated that the contact

inhibition ability of nontumor cells was not silenced, and hence the growth of nontumor cells was not inhibited

by these substances. The number of no-transfer control cells was higher than that in the transfer group, possibly

because the A549 condition medium was not suitable for the growth of nontumor cells. Based on the results

of reduced magnetic inhibition after suspension cell dispersion or cluster separation, it was speculated that the

substrates might be related to intercellular communication and cell contact inhibition. The intercellular signal

increased and the contact inhibition disappeared when cells were suspended and separated or clumps were too

far apart. Hence, the cells secreted one or more substrates, and the low-frequency magnetic fields interacted with

cells and substrates to specifically inhibit tumor cells.

The relationship between relevant substances, conditioned media, and contact inhibition needs further experi

mental verification.

 

4. Our low-frequency magnetic field in vitro experiments had a common point that the magnetic inhibition

efficiency increased with the increase in exposure time when the cells clustered and the culture environment

was stable (infusion group conditioned medium), which was consistent with most current reports. Mean

while, most reports also suggested that the magnetic field intensity positively correlated with the magnetic

efficiency. Hence, the magnetic suppression efficiency positively correlated with the exposure time of mag

netic field intensity2,3,19,20.

 

For the low-frequency field exposure, Trypan blue staining revealed no obvious cell staining and no significant

difference in cell activity, which might be related to the fact that cell death was not observed due to direct stain

ing after the treatment. It indicated that the magnetic field did not directly kill cells, but continuously induced

apoptosis; therefore, the cells were not stained with Trypan blue. Other reports showed that the intracellular

caspase-3 activity was upregulated and the membrane integrity decreased after magnetic field exposure2,3 . Some

reports revealed that some tumor cells had staining efficiency19, which might be related to cell type and detection

time selection. Annexin V staining also showed a progressive magnetic inhibition. However, the effect varied

by cell type2 .

 

The specific effect of magnetic fields on signaling in the cellular environment is still unclear. The signaling

pathway involved in the magnetic field may be related to contact inhibition and epigenetic inheritance. Prompted

by cellular signals1,22,27–29, we presume the presence of electrical signals between cells and calcium ions, which

are the second messenger associated with cell division. Several fluorescent probe experiments showed that the

change in membrane potential was related to the exposure to the magnetic field, and the membrane potential

reaction was different between adherent and suspended cells. The membrane potential of adherent cells tended to

be hyperpolarized under the magnetic field. The membrane potential of clustered suspended tumor cells tended

to depolarize under the magnetic field. This might be related to the cell growth and the associated intercellular

communication response under the magnetic field. As shown in Fig. 3c and e, suspended and isolated Raji cells

grew faster under the magnetic field. The findings of Yong Zhou's team27 explained this phenomenon. The cell

growth rate increased during depolarization, which involved the nanoaggregation of k-Ras switches on the

membrane surface. The suspended tumor cells dispersed with each other, the intercellular signaling by certain

substances increased, and the magnetic field−induced cell contact inhibition disappeared. Meanwhile, the cell

membrane potential of suspended tumor cells was depolarized, which accelerated cell growth, and the inhibition

decreased or disappeared. The intracellular calcium signal and the membrane potential with the change in the

magnetic field were relatively synchronized (except A549). We hypothesized that the calcium ion concentration

was associated with the membrane potential on magnetic field exposure. In addition, only 293 T cells showed

significant differences in the calcium ion concentration when exposed to the magnetic field. This was possibly

related to the hypermethylation of the tumor-associated calcium signaling network30. At present, the relationship

between calcium ion concentration and membrane potential and the inhibition signal of the magnetic field is not

clear. In addition, the intracellular pH of A549 cells increased under the magnetic field. This might be related to

the fact that A549 cells were more sensitive to the magnetic field. Also, this might be the reason why the calcium

change in A549 cells was not synchronized with the membrane potential.

 

This study had certain limitations. In the experiments on suspended tumor cells, we used a pipette to destroy

the cluster structure, and we could not keep the cells separated all the time during magnetic field exposure. In

addition, we could not maintain cell aggregation during the transfer to larger containers every 3 days. In detect

ing ions and membrane potential, we knew that the membrane potential was related to magnetic field exposure.

However, the kit could only measure the state of a certain period, and hence synchronous real-time detection is

needed to clarify the specific relationship. Moreover, the changes in the membrane potential also indicated the

changes in intracellular signals. Therefore, it was speculated that the membrane potential could be used as the

means to verify the inhibitory effect of the magnetic field on tumor cells.

 

Combining the aforementioned four key properties of the inhibitory effect of the magnetic field on cells

might serve as a good adjuvant anticancer modality. At present, magnetic field therapy has gained increasing

attention. Moreover, the universality of cell signals with magnetic field exposure and the broad spectrum of

tumors render it an excellent methodology for treatment and prognosis. Our findings, along with other reports,

further revealed the potential of magnetic field therapy. Our next study will focus on the mechanisms involved

in magnetic fields, starting with substrates.

 

                                      Methods

Test kit.  A Calbryte 520 AM calcium probe kit (item no. 20650) was purchased from AATBioquest (USA).

Hanks' buffer with 20 mM HEPES (item no. 20011) and DiBAC4 (3) membrane potential fluorescent probe

(item no. 21411) were also purchased from AATBioquest. A sodium ion fluorescent probe SBFI (item no. 18764)

was purchased from Cayman Company (USA). A potassium ion fluorescent probe PBFI (item no. 21602) was

purchased from the Cayman Company. Pluronic F-127 (ST501-10G) was purchased from Beyotime Biotechnol

ogy.

 

Cell culture. 293 T cells, Hepg2 cells, and A549 cells were obtained from the Cell Bank of the Chinese Acad

emy of Sciences (Shanghai, China). The cell lines were cultured in DMEM (Biological Industries, Israel) with

10% fetal bovine serum (FBS, ExCell Bio, China) and 1% penicillin–streptomycin (P/S, Industries, Israel). The

cells were incubated at 37 °C in the presence of 5% CO2. All operations were conducted inside the vertical-flow

clean bench.

Raji cells were cultured in the RPMI1640 medium (Biological Industries) with 10% FBS and 1% P/S. The

cells were incubated at 37 °C in the presence of 5% CO2. All operations were conducted inside the vertical-flow

clean bench.

 

Magnetic field exposure and characteristics. The self-made magnetic field generator converted elec

trical signals into magnetic field signals through an enameled copper wire (Fig. 6, Fig. S4). The shell was made

of acrylonitrile butadiene styrene plastic material with specifications: 450×230×25 (L×D×H, mm3 ). The mag

netic field output could be changed by adjusting the frequency and amplitude of the power supply voltage of

the equipment to the magnetic field–generating device. The magnetic field–generating device was placed in an

incubator, and the magnetic field was measured with a Gauss meter (TES 1393; TES Electrical Electronic Corp,

Taiwan). The cells in the nonexposed group were placed in the same incubator (Thermo Scientific, USA). The

direction of the magnetic field was perpendicular to the magnetic field generator. During the whole experiment,

the intensity of the stray magnetic field in each incubator was less than 0.02 mT (0–0.02 mT), and the tempera

ture was adjusted to 37±0.18 °C.

 

Cell counting. Adherent cells: The supernatant was collected in a centrifuge tube and washed twice with

normal saline. The cleaned supernatant was extracted, added to the centrifuge tube, and then digested with

Figure 6. Magnetic field exposure system. (a) Schematic diagram of the magnetic field exposure system. Details

are described in the Methods section. (b) Magnetic field waveform.

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