Cell Biology International 2002, Vol. 26, No.

7, 599–603
doi:10.1006/cbir.2002.0883, available online at http://www.idealibrary.com on
EXTREMELY LOW FREQUENCY (ELF) PULSED-GRADIENT MAGNETIC FIELDS
INHIBIT MALIGNANT TUMOUR GROWTH AT DIFFERENT BIOLOGICAL LEVELS
XINCHEN ZHANG
1
, HUSHENG ZHANG
1
*, CONGYI ZHENG
2
, CHAOYANG LI
2
, XINSONG ZHANG
3
and WEI XIONG
4
1
Biomedical Physics Unit, Department of Physics, Wuhan University, Wuhan 430072, China;
2
College of Life
Science, Wuhan University, Wuhan 430072, China;
3
Business School, Wuhan University, Wuhan 430072 China;
4
Institute of System Engineering, Huazhong University of Science & Technology, Wuhan 430074, China
Received 19 April 2001; accepted 17 January 2002
Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of
0.6–2.0 T, gradient of 10–100 T · M
1
, pulse width of 20–200 ms and frequency of 0.16–1.34 Hz
treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at
different hierarchical levels, from organism, organ, to tissue, and down to cell and macro-
molecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis,
preventing a supply developing to the tumour. The growth of sarcomas might be amenable to
such new method of treatment. 2002 Elsevier Science Ltd. All rights reserved.
Krxvo×ns: extremely low frequency (ELF) pulsed-gradient magnetic field; malignant tumour;
inhibitory; biological levels.
INTRODUCTION
Interactions between a malignant tumour and
the host can be affected by bioelectromangetic
phenomena. Many experiments have been carried
out to investigate the effect of magnetic fields on
tumour cell growth. It is reported that the incidence
of leukaemia in people residing near to power
transmission lines is higher (Poole, 1991; Chen,
1997). The frequency of fluctuating electromagnetic
fields around power transmission cables is 50 Hz,
whereas the varying frequency of pulse magnetic
fields approaches human physiology frequency of
1 Hz, or is a static magnetic field.
Two kinds of frequency range give completely
different biological effects. For example, Chang
et al. (1985) reported that pulsed magnetic field
(0.8 T, 22 ms, 1 Hz) inhibited the growth of S-180
sarcoma in mice, and have since used the same
magnetic field strength to treat patients with middle
and late-stage disease. Nine of 18 cases showed
good improvement, and nine were less well
inhibited (Chang et al., 1990). Since 1987, Wollin
(see Zhou, 1994) has treated effectively 50 cancer
cases with a Nd-Fe-B permanent magnet (0.4 T).
And we have then reported from electron micro-
scopic evidence the effects of ELF pulsed-gradient
magnetic field can not only inhibit the growth of
S-180 sarcoma in mice, but promote their oncolytic
ability of host immune cells (Zhang et al., 1995,
1997). We used the same magnetic field to treat six
patients with middle and late stage cancer in the
Tumour Department, Wuhan Center Hospital of
Guang-Zhou Military Region. Four cases were
highly effective, and two were moderately effective
(Zhang et al., 1994).
Apoptosis is a genetically regulated process.
Since the loss of balance between cell death and
division can be one of the factors leading to
carcinogenesis, much interest is shown in the
regulation of apoptosis (Fang et al., 1998; Silva
et al., 1996). We show here that an ELF Pulsed-
gradient magnetic field can induce apoptosis in
cancer cells, and that it may also block the devel-
opment of neovascularization required for tumour
supply.
*To whom correspondence should be addressed: E-mail:
hushengzhang@eLong.com
1065–6995/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved.
EXPERIMENTAL PROCEDURES
Treatment of mice in magnetic fields
Twenty Kunming mice , 2 months of age and
weighing (36–40 g), were kept in a single cage, thus
living conditions (food, water, temperature, light,
air, etc) were the same for all. They were randomly
divided into two groups, one being treated and the
other acting as its control. The mice were inocu-
lated in their right front legs with 110
6
S-180
sarcoma ascites cells. Sarcomas formed 4–5 days
after inoculation.
The treated mice were put in plexiglass boxes
with air holes, and located on the magnetic coil
(10 cm in diameter) of a customized (ELF) pulsed-
gradient magnetic field generator. When pulsed
electric current passed through the coil, the mag-
netic field had a maximum intensity of 0.6–2.0 T,
with a gradient of 10–100 T per metre, and the
pulse width was 20–200 ms with a frequency of
0.16–1.34 Hz being generated. The treated animals
were exposed to this magnetic field for 15 min
per day at room temperature 1826C, while no
treatment was applied to the control samples.
Twenty-eight days later, the mice were killed by
cervical dislocation, and the sarcomas cut out and
weighed. Sarcoma specimens were prepared for the
following examinations.
Optical sections and observation
Sarcomas with the volume of 1 cm
3
were fixed
in 10% formalin, dehydrated through graded
alcohols, brightened with ditoluene and soaked in
paraffin wax in an incubator at 60C. They were
finally embedded in paraffin wax at room tempera-
ture. Sections cut at 3–4 m were stained with
H & E. They were examined microscopically at 100
and 300 magnifications.
Transmission electron microscope (TEM) sections
and observation
Specimens of sarcomas of 1 mm
3
were fixed,
washed, post-fixed, dehydrated, and embedded
conventionally before being cut on an ultramicro-
tome at 50 nm. The sections were double-stained
with lead citrate and uranyl acetone before being
observed in a HU-11A TEM at 50 kV accelerating
voltage.
Detecting apoptosis through in situ
nick-end-labelling
Terminal deoxynucleotidyl transferase (TdT)-
mediated nick-end-labelling (TUNEL) was per-
formed with the ApopAlert DNA Fragmentation
Assay Kit, as instructed by its user manual
(CLONTECH, U.S.A.). Briefly, formalin-fixed,
paraffin-embedded cancer tissue sections were
mounted on glass slides (Ben-Sasson et al., 1995).
The paraffin wax was removed by immersing slides
in fresh xylene followed by 100% ethanol. Slides
were rehydrated through descending concen-
trations of ethanol (100%, 95%, 85%, 70%, 50%),
fixed in 4% formaldehyde/PBS, washed with PBS,
and treated with proteinase K. Subsequently, the
slides were incubated with buffer containing
fluorescein-dUTP and TdT enzyme, and the cells
stained with propidium iodide (PI), followed by a
final wash in PBS. The slides were examined as
soon as possible thereafter. Apoptotic cells exhib-
ited strong green nuclear fluorescence using a stan-
dard fluorescein filter of 52020 nm. All cells
staining with PI exhibited strong red cytoplasmic
fluorescence when viewed at 620 nm (Fang,
1998).
RESULTS AND DISCUSSION
Mice
The statistical results show that the mean weights
of the treated mice increased 5 d after the experi-
ment, while the mean weights of controls
decreased, indicating that the treated samples are in
better body conditions than the control ones. A
Table 1.
Comparison of weights of mice and sarcomas of treated and control groups
Groups Weight of the mice (XSD)/g Weight sarcomas
(SD)/g
0 day 5 day 10 day 15 day 20 day
Treated 38.103.50 42.093.24 43.703.57 46.115.66 50.388.59
a
1.400.81
b
Control 37.532.69 41.042.62 38.683.37 38.044.67 36.795.18 2.450.95
a
P<0.05;
b
P<0.01. X, Mean value; SD, standard difference.
600 Cell Biology International, Vol. 26, No. 7, 2002
statistical difference appeared within 20 days
(Table 1).
Organs and tissues
Sarcomas from treated mice were smaller and
harder than those from controls (Table 1). They
were more easily cut off from the surrounding
tissues. Their surfaces were covered with a viscid
capsular membrane (Fig. 1(a)). Sarcomas in
the control group were much larger and softer.
Lacking an intact viscid membrane, they seemed
to be joined tightly with the surrounding tissues
(Fig. 2(a)).
Endothelial cells of the blood vessel were swollen
in the treated mice tumours (Fig. 3), which seemed
Fig. 1. Treated sample, No. 2, 1.0 g. (A) Hard sarcoma
with the surface covered with viscid membrane. 2.5. (B)
Detection of apoptotic cancer cell TUNEL. Green shows
apoptotic cells, which make up 19.3% of the total. 200.
(C) Left: heterochromatin in a nucleus of cancer cell condensed
and coagulated together along the nuclear membrane. Right:
expanded endoplasmic reticulum. 7200.
Fig. 2. Control sample, No. 16, 3.3 g. (A) Softer sarcoma
joined tightly to its surroundings. 2.5. (B) Apoptotic cells
which make up 4.2% of the total. 200. (C) Left: large
nucleus of a cancer cell. Right: well-developed surface
endoplasmic reticulum. 7200.
Cell Biology International, Vol. 26, No. 7, 2002 601
to occlude the blood vessels, and therefore might
have shut off oxygen and nutrition supplies (Zhang
et al., 2000)
Cells
Compared with controls, it is found that the mito-
chondria and RER expanded in response to the
ELF treatment, indicating that the magnetic field
may have affected cell metabolism. It is normally
accepted that poorly differentiated cancer cells
have high N/P ratios, more heteromorphic nuclei,
are often multinucleolate, and these features relate
to rapid growth. N/P ratios were an indicator of the
malignancy of the cancer cells. Our microscopic
data indicate that the exposed sarcoma cells had
rounder nuclei, and that the volume fraction of
nucleus and N/P decreased (cf. Zhang et al., 1997).
These indicate that the magnetic field lower the
‘degree of malignancy’ of sarcoma cells and inhibits
their rapid and heteromorphic growth.
Cancer cells in the treated group showed some of
the morphological properties associated with pro-
grammed cell death (PCD). Cancer cell contacts
were looser and more divorced from adjacent cells.
Their heterochromatin condensed and coagulated
together along the nuclear membrane (Fig. 1(c)).
The endoplasmic reticulum seemed to be dilated
and fused with the cellular membrane. Many apop-
totic bodies, packed with cellular membrane debris,
appeared and were probably being digested by
lymphocytes and macrophages.
Extensive DNA degradation is characteristic in
the early stages of apoptosis. Cleavage of the DNA
may yield double-stranded fragments with 3-OH
termini as well as single strands. The free 3-ends, in
DNA can be labelled with DIG-dUTP by terminal
deoxynucleotidyl transferase (TdT), with the incor-
porated nucleotides being detected in a second
incubation step with an anti-DIG antibody conju-
gated with fluorescein. The immuno-complex has
an emission wavelength of 523 nm (green light)
when excited at 494 nm. Labelled apoptotic cells,
counted under fluorescence microscopy, in treated
sarcomas were greater on number than in the
controls (Fig. 1(b) and 2(b)).
The phenomenon of immune cells, including
lymphocytes and phagocytes, accumulating around
cancer cells to destroy and digest them was seen
more prominently in sarcomas exposed to the
magnetic field.
Macromolecules
We observed a decrease of DNA content by
Feulgen staining which indicated that magnetic
field can block DNA replication and mitosis of
sarcoma cells (Zhang et al., 1997). It is found that
a decrease of the mitotic phases of carcoma cells is
due to exposure (Zhang et al., 1995).
ELF pulsed-gradient magnetic field may be able
to inhibit the growth and division of cancer cell and
enhance the host cellular immune response. How
the low frequency pulsed-gradient magnetic field
induces apoptosis of cancer cell and blocks new
blood vessel development remains unknown, but it
nevertheless has been found that this could be a
new method for the treatment of cancer, although
clearly further research on the mechanism is much
needed.
ACKNOWLEDGEMENTS
The authors thank Prof. Li Tongping of the Patho-
logical Unit of Fundamental Medical School,
Tongi Medical University, for his kind instruction
and help. This work was supported by research
grants from the Natural Science Foundation of
China and the Ministry of Science and Technology
of China.
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SD. with a gradient of 10–100 T per metre. X. Briefly. standard difference.69 P<0. and the sarcomas cut out and weighed. They were finally embedded in paraffin wax at room temperature. the slides were incubated with buffer containing fluorescein-dUTP and TdT enzyme. 26. light.09 41. The treated animals were exposed to this magnetic field for 15 min per day at room temperature 1826 C. Comparison of weights of mice and sarcomas of treated and control groups Groups 0 day Treated Control a Weight of the mice (X 5 day 42. 2 months of age and weighing (36–40 g).59a 5. 95%. 70%. while no treatment was applied to the control samples. Sections cut at 3–4 m were stained with H & E.40 2. A . Optical sections and observation Sarcomas with the volume of 1 cm3 were fixed in 10% formalin.68 3. Twenty-eight days later.6–2. The slides were examined as soon as possible thereafter. When pulsed electric current passed through the coil. paraffin-embedded cancer tissue sections were mounted on glass slides (Ben-Sasson et al. etc) were the same for all. The treated mice were put in plexiglass boxes with air holes.62 10 day 43.10 37.79 8. were kept in a single cage. and embedded conventionally before being cut on an ultramicrotome at 50 nm. Slides were rehydrated through descending concentrations of ethanol (100%. while the mean weights of controls decreased. and located on the magnetic coil (10 cm in diameter) of a customized (ELF) pulsedgradient magnetic field generator. and the cells stained with propidium iodide (PI). All cells staining with PI exhibited strong red cytoplasmic fluorescence when viewed at 620 nm (Fang.81b 0. Detecting apoptosis through in situ nick-end-labelling Terminal deoxynucleotidyl transferase (TdT)mediated nick-end-labelling (TUNEL) was performed with the ApopAlert DNA Fragmentation Assay Kit.67 20 day 50.95 38. The paraffin wax was removed by immersing slides in fresh xylene followed by 100% ethanol.01.. Mean value.04 3.57 3.S.50 2.). followed by a final wash in PBS. as instructed by its user manual (CLONTECH. post-fixed.04 5. Vol.24 2. 1998). indicating that the treated samples are in better body conditions than the control ones. dehydrated through graded alcohols.18 Weight sarcomas ( SD)/g 1. The mice were inoculated in their right front legs with 1 106 S-180 sarcoma ascites cells. thus living conditions (food. 7. 50%). water. and treated with proteinase K.600 Cell Biology International.0 T.38 36.70 38. U. They were randomly divided into two groups. air. formalin-fixed.34 Hz being generated. RESULTS AND DISCUSSION Mice The statistical results show that the mean weights of the treated mice increased 5 d after the experiment. dehydrated. Subsequently. the mice were killed by cervical dislocation.37 SD)/g 15 day 46. They were examined microscopically at 100 and 300 magnifications.05. Sarcomas formed 4–5 days after inoculation. bP<0. temperature. fixed in 4% formaldehyde/PBS. one being treated and the other acting as its control. The sections were double-stained with lead citrate and uranyl acetone before being observed in a HU-11A TEM at 50 kV accelerating voltage. Apoptotic cells exhibited strong green nuclear fluorescence using a standard fluorescein filter of 520 20 nm.66 4. 1995). Transmission electron microscope (TEM) sections and observation Specimens of sarcomas of 1 mm3 were fixed. the magnetic field had a maximum intensity of 0. EXPERIMENTAL PROCEDURES Treatment of mice in magnetic fields Twenty Kunming mice .53 3. brightened with ditoluene and soaked in paraffin wax in an incubator at 60 C. 85%. Sarcoma specimens were prepared for the following examinations.45 0.A. 2002 Table 1.11 38. washed. and the pulse width was 20–200 ms with a frequency of 0. No.16–1. washed with PBS.

which seemed .3 g. 2. 1. they seemed to be joined tightly with the surrounding tissues (Fig. (B) Detection of apoptotic cancer cell TUNEL. 1(a)). Right: expanded endoplasmic reticulum.Cell Biology International. Their surfaces were covered with a viscid capsular membrane (Fig. Fig. 7. (A) Softer sarcoma joined tightly to its surroundings. They were more easily cut off from the surrounding tissues. 7200. No. 2. 3. No. (A) Hard sarcoma with the surface covered with viscid membrane. 2. 200. 3). (C) Left: large nucleus of a cancer cell. Endothelial cells of the blood vessel were swollen in the treated mice tumours (Fig. 2002 601 Fig.3% of the total.5. 2(a)).5. Right: well-developed surface endoplasmic reticulum. Sarcomas in the control group were much larger and softer. Green shows apoptotic cells. 26. 7200.0 g. Control sample. Vol. No. (C) Left: heterochromatin in a nucleus of cancer cell condensed and coagulated together along the nuclear membrane. statistical difference appeared within 20 days (Table 1). 200. 1. 16. 2.2% of the total. Organs and tissues Sarcomas from treated mice were smaller and harder than those from controls (Table 1). which make up 19. Lacking an intact viscid membrane. Treated sample. (B) Apoptotic cells which make up 4.

Cancer cell contacts were looser and more divorced from adjacent cells. Our microscopic data indicate that the exposed sarcoma cells had rounder nuclei. 1995). Extensive DNA degradation is characteristic in the early stages of apoptosis. How the low frequency pulsed-gradient magnetic field induces apoptosis of cancer cell and blocks new blood vessel development remains unknown. accumulating around cancer cells to destroy and digest them was seen more prominently in sarcomas exposed to the magnetic field. N/P ratios were an indicator of the malignancy of the cancer cells. and therefore might have shut off oxygen and nutrition supplies (Zhang et al. 7. to occlude the blood vessels. ELF pulsed-gradient magnetic field may be able to inhibit the growth and division of cancer cell and enhance the host cellular immune response. This work was supported by research grants from the Natural Science Foundation of China and the Ministry of Science and Technology of China. Zhang et al. 1. It is found that a decrease of the mitotic phases of carcoma cells is due to exposure (Zhang et al. but it nevertheless has been found that this could be a new method for the treatment of cancer. These indicate that the magnetic field lower the ‘degree of malignancy’ of sarcoma cells and inhibits their rapid and heteromorphic growth. The phenomenon of immune cells. Cleavage of the DNA may yield double-stranded fragments with 3 -OH termini as well as single strands. Vol. although clearly further research on the mechanism is much needed. and these features relate to rapid growth. in treated sarcomas were greater on number than in the controls (Fig. thus the blood vessel is blocked..5 g. 1985. appeared and were probably being digested by lymphocytes and macrophages. The endoplasmic reticulum seemed to be dilated and fused with the cellular membrane. for his kind instruction and help. The free 3 -ends. Cancer cells in the treated group showed some of the morphological properties associated with programmed cell death (PCD). 2002 Fig. including lymphocytes and phagocytes. indicating that the magnetic field may have affected cell metabolism. 1(c)). No.. packed with cellular membrane debris. 26. et al. are often multinucleolate. more heteromorphic nuclei. Macromolecules We observed a decrease of DNA content by Feulgen staining which indicated that magnetic field can block DNA replication and mitosis of sarcoma cells (Zhang et al. The endothelium cell of blood vessel are swelled by magnetic field. Treated sample. Labelled apoptotic cells.. P Y. 1997). with the incorporated nucleotides being detected in a second incubation step with an anti-DIG antibody conjugated with fluorescein. Their heterochromatin condensed and coagulated together along the nuclear membrane (Fig. Tongi Medical University. . 1997). and that the volume fraction of nucleus and N/P decreased (cf. 2000) Cells Compared with controls. It is normally accepted that poorly differentiated cancer cells have high N/P ratios. 1(b) and 2(b)). Chinese Journal of Physics Medicine 7: 169–170 (in Chinese). 5000. 0. No. Li Tongping of the Pathological Unit of Fundamental Medical School. L G. 3. Many apoptotic bodies.. REFERENCES C H. The immuno-complex has an emission wavelength of 523 nm (green light) when excited at 494 nm. counted under fluorescence microscopy. ACKNOWLEDGEMENTS The authors thank Prof.. in DNA can be labelled with DIG-dUTP by terminal deoxynucleotidyl transferase (TdT). Experimental observation on the effect of magnetic field on S-180 sarcomas in mice. it is found that the mitochondria and RER expanded in response to the ELF treatment.602 Cell Biology International.

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