Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanismJie Sun1,2,3, YingyingTong1,2,3, Yu Jia1,2,3, Xu Jia1,2,3, Hua Wang4 , Yang Chen1,2,3, JiaminWu5 ,
Weiyang Jin5 , Zheng Ma6 , Kai Cao6 , Xiangdong Li6 , Zhonglin Chen6 & GuanghuaYang1,2,3*
Low-frequency magnetic fields exert a significant inhibitory effect on tumor growth and have been
developed as a therapeutic modality. However, the effect of a low-frequency magnetic field on
the interaction between cells is still poorly understood. This study aimed to preliminarily evaluate
the direct effect of magnetic field ditectely on cultured cells and indirect effect mediated by cell
environment (conditioned medium). 293T cells, Hepg2 cells, A549 cells have been cultured at
37 ± 0.18 °C in presence of an extremely low-frequency magnetic field of 20 Hz, 5-mT. The adherent
tumor cells were more sensitive to magnetic field inhibition in the original environment (conditioned
medium) with adherence inhibition rate for Hepg2 and A549 estimated at 18% and 30% respectively.
The inhibition effect was suppressed when the suspended cells separated or clump density at a low
density. The nontumor cell lines showed no inhibitory effect on exposure to a low-frequency magnetic
field. The intracellular ion fluorescence (IIF) showed that the magnetic field significantly altered
the membrane potential, indicating hyperpolarization of the adherent cells (ΔIIF 293T cells: − 25%,
ΔIIF Hepg2 cells: − 20% and ΔIIF A549 cells: − 13%) and depolarization of the suspended cells (ΔIIF
Raji cells: + 9%). In addition, the conditioned media collected after magnetic field exposure acted on
unexposed tumor cells and caused inhibition. Our findings might provide a basis for the mechanism of
magnetic field interaction between cells and cell environment in the future.
Low-frequency magnetic fields exert noninvasive, nonionizing, and nonthermal effects on cells and tissues.
They enhance the cellular oxidative stress response and regulate the apoptotic signaling pathway, changing the
intracellular Ca2+ concentration to induce apoptosis1–3 . They are widely used to treat tumors and neuropsychi
atric and bone diseases. In vivo studies in this field have shown that low-frequency magnetic fields inhibit the
proliferation of tumor cells and prolong their survival4–11.
In most reports on using magnetic fields as a combination therapy, extremely low-frequency magnetic fields
enhance the efficacy of antitumor drugs12–15. A combination of an extremely low-frequency magnetic field
with paclitaxel in treating mouse cancer revealed that the magnetic field increased the execution lethality of
paclitaxel16. The cell membrane permeability was altered, and the therapeutic effect of cisplatin was significantly
enhanced at an extremely low-frequency magnetic field of 10 mT combined with cisplatin17. However, Gellrich
found that the low-frequency magnetic field could not enhance the therapeutic effect of cetuximab, which might
be related to the conformational change in the molecular surface receptor18.
In most in vitro experiments, the low-frequency magnetic field showed a significant inhibitory effect on tumor
cells2,3,19–23 and did not affect the growth of normal cells2,24. A report found that the magnetic field affected the
surface of the tumor membrane, thus influencing tumor proliferation25. However, some reports showed that the
proliferation of tumor cells slightly increased under the low-frequency magnetic field26.
1International Research Center for Biological Sciences, Ministry of Science and Technology, Shanghai Ocean
University, Shanghai 201306, China. 2 National Aquatic Animal Pathogen Collection Center, Shanghai Ocean
University, Shanghai 201306, China. 3 Aquatic Animal Genetics and Breeding Center, Shanghai Ocean University,
Shanghai 201306, China. 4 Shanghai Telebio Biomedical Co., Ltd, Shanghai, China. 5 Zhejiang Huayi Health Industry
Development Co., Ltd, Hangzhou, China. 6 Huisi Anpu Medical System Co., Ltd, Qinhuangdao, China.
*email:ghyang119@163.com
At present, it is believed that magnetic fields can significantly inhibit tumor growth, and the inhibitory effect
has a positive correlation with time and intensity. Meanwhile, the production of reactive oxygen species (ROS)
is an inevitable phenomenon considered to be the key to the inhibitory effect of the magnetic field3 . However,
the exact mechanism is unclear. In the development of antineoplastic therapies, the inhibitory effect of the mag
netic field on tumor growth is a significant attribute to the clinical performance of many existing technologies.
Many experiments have been conducted on the differences in magnetic field settings, but little research has
been done on the effect of the difference in the magnetic field on the tumor environment and the possible inhibi
tion mechanism, except ROS. In this study based on the effect of a magnetic field on the intercellular environment
and intercellular structure (the form of natural contact between cells and the form of human interference), the
cells were cultured in vitro. The study found that the state of intercellular aggregation was a necessary phenom
enon for magnetic inhibition. At the same time, during cell proliferation, one or several related substrates were
released in the conditioned medium, which might act together with the magnetic field to achieve the effect of
magnetic field inhibition.
In this experiment, a magnetic field of 5 mT and 20 Hz was used as the sole background. In previous experi
ments, the magnetic field intensity was not fixed, and it often did not directly contact cells or could not be placed
in an incubator. The simple magnetic field generator designed in this study could be in direct contact with cells
and placed in an incubator under stable conditions of temperature and CO2. Our magnetic field generator also
had disadvantages, that is, when a magnetic field was generated, it also generated heat. Based on the intensity
design of Crocetti19, a magnetic field generator was designed to stabilize the heat through heat dissipation.
Results：Magnetic field inhibited the adherent tumor cells, which was influenced by the difference in
the culture environment (conditioned medium). The treatment groups were divided into two groups
before exposure. Infusion group: Prior to daily exposure, 500 µL of the fresh medium was slowly added with
a pipettor to the pore wall. The conditioned culture medium was a mixture of various substances (medium
incubated overnight after passage and lamination) and a fresh medium for exponential growth. Change group:
Prior to daily exposure, a pipette was applied to the pore wall to remove most of the medium (almost all), which
was replaced with a medium of the same volume as the "infusion" group. The conditioned medium was com
pletely fresh with no or minimal secretions. The difference between the two groups was the composition of the
conditioned medium: the composition in the "infusion group" was more complex, while the composition in the
"change group" was closer to that of the unused medium. Normal human renal epithelial cells 293 T, human liver
cancer cells Hepg2, and human nonsmall-cell lung cancer cells A549 were processed independently through
culture medium "infusion" or "change" and exposed to the 5-mT intensity of the magnetic field for 2 h each day
for a total of 3 days. The initial number of all cells was 2× 105 . Figure 1a shows that the nontumor cell line 293 T
was not inhibited by the magnetic field in the "infusion group" and the "change group". Figures 1b and c shows
that the number of Hepg2 and A549 cells exposed to the magnetic field was significantly lower than that of cells
in the unexposed control group. Both tumor cell lines Hepg2 and A549 were inhibited by the magnetic field
(the highest inhibition rate of Hepg2 was about 18%, and that of A549 was about 30%). The tumor cells in the
"infusion group" showed inhibition on day 1, while the cells in the "change group" showed no significant inhibi
tion. The inhibition trend in the "infusion group" was significantly stronger than that in the "change group". The
inhibitory effect in the "infusion group" (A549) positively correlated with exposure duration (Fig. 1d). These
results indicated that the tumor cells were more sensitive to the magnetic field in the conditioned medium
(microenvironment) modified by autocrine and paracrine signals.
Spontaneous aggregation of suspended tumor cells was destroyed, and magnetic inhibition
disappeared. We investigated whether a nutrient loss in the conditioned medium, excessive cell density,
and interference with magnetic field inhibition by trypsin could affect the longer duration of magnetic inhibi
tion. In particular, we performed the experiment using suspended lymphoma Raji cells. In natural culture, the
suspended cells spontaneously gathered into clusters. The structure of such clusters was inevitably destroyed
when the centrifuged cells were replaced with a conditioned medium in the change group. We also destroyed the
cluster structure in the infusion group to ensure the consistency of experimental conditions. The suspended cells
were infused or changed with the culture medium and exposed to a 5-mT magnetic field for 2 h daily for 6 days.
Infusion group: Prior to daily exposure, 500 µL of the fresh medium was slowly added with a pipettor to the pore
wall, and the cells were blown with a pipette gun to separate them in suspension and destroy the agglomerative
structure. Change group: Prior to daily exposure, the cell suspension was aspirated and centrifuged at 1200 rpm
to remove the supernatant, which was then replaced with a medium of the same volume as the "infusion" group.
On day 3, the conditioned medium was completely replaced in the infusion group, while transferring the cells
from both groups to larger containers. The suspended tumor cells were cultured in vitro without trypsin and
easily transferred to larger containers, avoiding trypsin damage. Under such conditions, neither group showed
significant inhibition compared with the no-exposure control group.
The initial number of all cells was 2× 105 . Figure 2a shows no significant difference in the number of cells in
the "infusion group" and "change group" after 6 days of magnetic field exposure compared with the no-exposure
control group. Figure 2b shows that the magnetic field inhibition was not obvious after the aggregation structure
disappeared.
As shown in Fig. 2a, the number of cells in the "infusion group" was significantly higher than that in the
"change group" on day 4 (after replacing the large container). The "infusion group" environment was more suit
able for the growth of suspended tumor cells.
Figure 1. Difference in the environment (conditioned medium) before exposure affected the inhibitory effect
of the magnetic field on adherent cells. (a) No significant difference was found in the number of 293 T cells in
3 days. (b) Number of Hepg2 cells in the unexposed and exposed groups was significantly different. (c) Number
of A549 cells in the unexposed and exposed groups was significantly different. (d) Cell inhibition curve. The
cell inhibition rate in the infusion group was more obvious than that in the change group, and the normal cell
inhibition rate was not of statistical significance (* P<0.05, versus the no-exposure control group).
Figure 2c shows suspended cells in separate and natural suspended states. It was seen that most cells were
single-celled instead of multi-celled clusters after the destruction of the cluster structure.