- Email: [email protected]
Influence of electromagnetic fields on morphology and mitochondrial activity of breast cancer cell line MCF7
127
Bioelectrochemistry and Bioenergetics, 30 (1993) 127-132 Elsevier Sequoia S.A ., Lausanne
JEC BB 02034
Influence of electromagnetic fields on morphology and mitochondrial activity of breast cancer cell line MCF7 S. Johann ", T. Lederer, S . Mikorey, W. Kraus and G . Bliimel Institut f4 r Experimentelle Chirurgie der TU Miinchen, Ismaninger Str. 22, 8000 Miinchen 80 (Germany)
Abstract Cells of the breast cancer cell line MCF7 were exposed to electromagnetic fields (EMF) . A sinusoidal signal (12 h/day) was generated by a function generator with a frequency of 20 Hz and a maximum magnetic induction of 5 .3 mT. After 6 days EMF subjected cells showed a significant increase in mitochondrial activity as shown by MTT-assay . At the same time EMF exposed cells investigated by scanning electron microscopy indicated intensified membrane ruffling and pronounced microvilli development compared with control cells .
INTRODUCTION
In numerous clinical studies electromagnetic fields (EMF) have proved to have beneficial effects on pseudarthrosis [1], and on aseptic loosening of total hip prostheses [2] . Moreover, their impact on wound healing [3], on osteoporosis [4] and on tumor growth [5] is under close investigation . Despite its common clinical use, little is known about the impact of EMFs at the cellular and/or molecular level . Controversial results have been obtained owing to the wide variety of EMF characteristics and to the use of different cell lines and culture conditions . A pulsing magnetic field is able both to stimulate or inhibit cellular DNA synthesis in Chinese hamster V79 cells, depending on the magnetic intensity rather than on the pulse frequency [6] . Low-frequency EMFs increased total protein synthesis and collagen accumulation in chicken fibroblasts, but no effect on cell proliferation was observed [7] . Growth characteristics and colony-forming efficiency in two mammalian tumor lines were not affected by EMFs [8] . However, EMFs stimulated the antitumor effect of the antiblastic drug mitomycin * To whom correspondence should be addressed . 0302-4598/93/$06 .00 © 1993 - Elsevier Sequoia S .A. All rights reserved
128
C on these cells . Similar results were communicated by Petrini et al . [9] reporting an increased activity of doxorubicine by EMFs. The fascinating aspects of modulating the proliferative capacity of cells by means of EMFs prompted us to quantify effects of intermittent EMF (6 h on, 6 h off) on the mitochondrial activity of the established human breast cancer cell line MCF7 . Morphological changes in cell appearance due to EMF exposure were documented by scanning electron microscopy (SEM). MATERIALS AND METHODS Electromagnetic field
A function generator (Magnetodyn M70S) was used to drive two solenoid coils, each of which could take up three 96-well plates . The experimental set-up is shown in Fig . 1 . A continuously sinusoidal signal was generated with a frequency of . 20 Hz and a maximum magnetic induction of 5 .3 mT . The distortion factor was less than 1% as measured with a gaussmeter (Bell 640 ) and a spectrum analyzer (HP 3582) . These signal shapes correspond to those used clinically to improve fracture and wound healing [10] . The coils were installed in an incubator at 37°C and were prevented from increasing the incubation temperature by a Haake cooling system with continuous temperature monitoring . Temperature differences between the control and experimental set-up were less than 0 .15°C during intermittent EMF application . Cells and culture conditions
The established human breast cancer cell line MCF7 (ATCC HTB 22) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
Temperature control in nutrient solution : 37 ' ± 0,3 'C
Cooling System Haake F3-CH
Function Generator Magnetodyn® M70 5
Fig . 1. Experimental set-up of the magnetic field in two solenoid coils P6-K
129
calf serum (FCS) and 1% L-glutamine, 1% sodium pyruvate, 1% streptomycin and 10000 IU penicillin/ml . The cells were incubated at 37°C in a humidified atmosphere of 5% CO 2. Cells were passaged twice a week . Cell viability was routinely determined using trypan blue dye exclusion . Prior to the first application of EMF, each sample was incubated for 6 h under the above mentioned conditions . MTT assay
The mitochondrial activity of MCF7 cells was measured using the ability of viable cells to transform tetrazolium salts into insoluble formazan [11] . Cells were trypsinized, washed with phosphate buffered saline (PBS) and adjusted to ,5 x 10 5 cells/ml . One hundred µl of diluted cell suspension was seeded in 96-well plates in concentrations of 1 x 10 2 to 5 x 10 4 /ml (twelvefold). Control and exposed cells were incubated for 2-14 days under standard conditions . At the end of each incubation period cells were immediately subjected to dimethyl-thiazolyl-diphenyltetrazolium bromide (MTT) assay for which 10 µl MTT in 5 mg/ml PBS was added to each well and cells were incubated for a further 4 h. Then, precipitated formazan was dissolved in 200 µl 0.04 M HCl and the amount of MTT-formazan was quantified in an ELISA reader by absorbance at 550 rim . Scanning electron microscopy
1 x 10 6 cells were placed into Leighton tubes (5 cm 2 growth area) and exposed to an intermittent EMF for three days . Cells were washed twice with PBS and were fixed with glutaraldehyde (1%) at 4°C for 16 h . Specimens were dehydrated through graded ethanols, and transferred to isoamylacetate, followed by criticalpoint-drying with liquid carbon dioxide. After coating with gold, specimens were observed under a scanning electron microscope (AMR Leitz 1200B) under an acceleration voltage of 15 kV . RESULTS
The MTT assay showed a significant increase in mitochondrial activity in cell populations that had been subjected to EMF for 14 days. The difference in mitochondrial activity between EMF-stimulated cells and control cells was clearly detectable (31%) when low numbers of cells (10 cells/well) had been seeded . Differences were less (17-21%) for initially higher numbers of cells (20-50 cells/well) . Starting with 100 cells/well the MTT reduction approached a plateau for both control and exposed cells (Fig . 2). Cells alternately exposed to EMF (3 days of intermittent EMF application followed by 3 days of culturing without EMF) showed a higher MTT reduction than control cells (without EMF), but a lower MTT reduction than cells with continuous 6 day EMF exposure . Regardless of the initial cell concentration there
130
0,10 100
200 500 initial cell count/ml
1,000
Fig . 2. Reduction of MTT in MCF7 cells incubated for 14 days and exposed to EMF (A) and omitting EMF (0) . Cell counts as seeded at the beginning of the experiment . Shown are the means of 36 measurements out of three independent experiments . * : p < 0.05 .
was a significant increase in MTT reduction in EMF-stimulated cells to unstimulated cells after 6 days exposure (Fig . 3) . SEM revealed morphological alteration of the cell surface due to EMF . Membrane ruffling and microvilli formation are more pronounced under EMF . Intercel-
0,0
100
500
1,000 2,Q00 initial cell count/well
5y000
Fig. 3. Determination of MTT reduction in MCF7 cells under the influence of EMF for 6 days (A), without the influence of EMF (0) and three days under EMF followed by three days without (x) . Shown are means of 60 measurements out of five independent experiments .
131
Fig. 4 . SEM image showing MCF7 control cells incubated for 72 h . Magnification 1920 X . lular junctions remained unaltered . The arrangement of villous projections was not affected by exposure to EMF (Figs. 4 and 5) . DISCUSSION AND CONCLUSION As shown by Mosmann [11] mitochondrial activity of the cell population correlates the number of cells . Therefore, at the end of each experiment mitochon-
Fig . 5 . SEM image showing MCF7 cells after exposure to EMF for 36 h (total incubation time 72 h) . Magnification 1920 X .
132
drial activities of the cell populations were determined by means of the MTT assay . Under the influence of EMF, MCF7 cells showed an increased MTT reduction compared with the control over a broad range of initial population densities . This effect was first detectable after 6 days of intermittent exposure and was even more pronounced after 14 days . This effect could first be detected after two days continuous exposure (data not shown) . Increased MTT reduction correlated to morphological changes in that the surface structure of MCF7 cells appeared more rough and uneven . EMF characteristics, culturing conditions, and the cell types used clearly demand further specifications as changes vary with different experimental settings . For example, Akamine et al . [121 reported on pulsed magnetic field treatment of embryonal carcinoma cells which showed regular arrangement and smoother surface then control cells. The positive effect of EMFs shown in clinical applications is also substantiated in the present paper . Nevertheless knowledge as to why EMFs may produce beneficial effects in vivo is still patchy . Future work should therefore be directed towards the understanding of the (molecular) mechanisms by which EMFs interact with cell replication and cell proliferation . REFERENCES 1 W . Kraus and F . Lechner, Munchner Med. Wochenschrift, 42 (1972) 1814 . 2 R. Ascherl, G . Blumel, F . Lechner, W. Kraus, Biochem . Bioenerg ., 14 (1985) 161 . 3 W. Mnhlbauer, Langenbecks Arch. Chir., 337 (1974) 637. 4 F . Tabrah, M . Hoffmeister, F . Gilbert, S . Batkin, C. Bassett, J. Bone Mineral Res., 5 (1990) 437. 5 E. Azavedo, Clin . Radiol., 43 (1991) 84 . 6 K . Takahashi, I . Kaneko, M . Date and E. Fukada, Experientia, 42 (1986) 185 . 7 J.C . Murray and R .W. Famdale, Biochim . Biophys . Acta, 838 (1985) 98 . 8 Y . Omoto, M . Hosokawa, M . Komatsumoto, T. Namieno, S . Nakajima, Y. Kubo and H . Kobayashi, Jpn. J. Cancer Res ., 81 (1990) 956 . 9 M. Petrini, L. Mattii, A. Sabbatini, G. Carulli, B. Grassi, R . Cadossi, G. Ronca and A. Conte, J. Bioelec., 9 (1990) 209 . 10 W. Kraus, Orthopade, 13 (1984) 78. 11 T. Mosmann, J. Immunol. Meth.,_65 (1983) 55 . 12 T . Akamine, H . Muramatsu, H. Hamada and T. Sakou, J. Cell. Physiol., 124 (1985) 247 .
Bioelectrochemistry and Bioenergetics, 30 (1993) 127-132 Elsevier Sequoia S.A ., Lausanne
JEC BB 02034
Influence of electromagnetic fields on morphology and mitochondrial activity of breast cancer cell line MCF7 S. Johann ", T. Lederer, S . Mikorey, W. Kraus and G . Bliimel Institut f4 r Experimentelle Chirurgie der TU Miinchen, Ismaninger Str. 22, 8000 Miinchen 80 (Germany)
Abstract Cells of the breast cancer cell line MCF7 were exposed to electromagnetic fields (EMF) . A sinusoidal signal (12 h/day) was generated by a function generator with a frequency of 20 Hz and a maximum magnetic induction of 5 .3 mT. After 6 days EMF subjected cells showed a significant increase in mitochondrial activity as shown by MTT-assay . At the same time EMF exposed cells investigated by scanning electron microscopy indicated intensified membrane ruffling and pronounced microvilli development compared with control cells .
INTRODUCTION
In numerous clinical studies electromagnetic fields (EMF) have proved to have beneficial effects on pseudarthrosis [1], and on aseptic loosening of total hip prostheses [2] . Moreover, their impact on wound healing [3], on osteoporosis [4] and on tumor growth [5] is under close investigation . Despite its common clinical use, little is known about the impact of EMFs at the cellular and/or molecular level . Controversial results have been obtained owing to the wide variety of EMF characteristics and to the use of different cell lines and culture conditions . A pulsing magnetic field is able both to stimulate or inhibit cellular DNA synthesis in Chinese hamster V79 cells, depending on the magnetic intensity rather than on the pulse frequency [6] . Low-frequency EMFs increased total protein synthesis and collagen accumulation in chicken fibroblasts, but no effect on cell proliferation was observed [7] . Growth characteristics and colony-forming efficiency in two mammalian tumor lines were not affected by EMFs [8] . However, EMFs stimulated the antitumor effect of the antiblastic drug mitomycin * To whom correspondence should be addressed . 0302-4598/93/$06 .00 © 1993 - Elsevier Sequoia S .A. All rights reserved
128
C on these cells . Similar results were communicated by Petrini et al . [9] reporting an increased activity of doxorubicine by EMFs. The fascinating aspects of modulating the proliferative capacity of cells by means of EMFs prompted us to quantify effects of intermittent EMF (6 h on, 6 h off) on the mitochondrial activity of the established human breast cancer cell line MCF7 . Morphological changes in cell appearance due to EMF exposure were documented by scanning electron microscopy (SEM). MATERIALS AND METHODS Electromagnetic field
A function generator (Magnetodyn M70S) was used to drive two solenoid coils, each of which could take up three 96-well plates . The experimental set-up is shown in Fig . 1 . A continuously sinusoidal signal was generated with a frequency of . 20 Hz and a maximum magnetic induction of 5 .3 mT . The distortion factor was less than 1% as measured with a gaussmeter (Bell 640 ) and a spectrum analyzer (HP 3582) . These signal shapes correspond to those used clinically to improve fracture and wound healing [10] . The coils were installed in an incubator at 37°C and were prevented from increasing the incubation temperature by a Haake cooling system with continuous temperature monitoring . Temperature differences between the control and experimental set-up were less than 0 .15°C during intermittent EMF application . Cells and culture conditions
The established human breast cancer cell line MCF7 (ATCC HTB 22) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
Temperature control in nutrient solution : 37 ' ± 0,3 'C
Cooling System Haake F3-CH
Function Generator Magnetodyn® M70 5
Fig . 1. Experimental set-up of the magnetic field in two solenoid coils P6-K
129
calf serum (FCS) and 1% L-glutamine, 1% sodium pyruvate, 1% streptomycin and 10000 IU penicillin/ml . The cells were incubated at 37°C in a humidified atmosphere of 5% CO 2. Cells were passaged twice a week . Cell viability was routinely determined using trypan blue dye exclusion . Prior to the first application of EMF, each sample was incubated for 6 h under the above mentioned conditions . MTT assay
The mitochondrial activity of MCF7 cells was measured using the ability of viable cells to transform tetrazolium salts into insoluble formazan [11] . Cells were trypsinized, washed with phosphate buffered saline (PBS) and adjusted to ,5 x 10 5 cells/ml . One hundred µl of diluted cell suspension was seeded in 96-well plates in concentrations of 1 x 10 2 to 5 x 10 4 /ml (twelvefold). Control and exposed cells were incubated for 2-14 days under standard conditions . At the end of each incubation period cells were immediately subjected to dimethyl-thiazolyl-diphenyltetrazolium bromide (MTT) assay for which 10 µl MTT in 5 mg/ml PBS was added to each well and cells were incubated for a further 4 h. Then, precipitated formazan was dissolved in 200 µl 0.04 M HCl and the amount of MTT-formazan was quantified in an ELISA reader by absorbance at 550 rim . Scanning electron microscopy
1 x 10 6 cells were placed into Leighton tubes (5 cm 2 growth area) and exposed to an intermittent EMF for three days . Cells were washed twice with PBS and were fixed with glutaraldehyde (1%) at 4°C for 16 h . Specimens were dehydrated through graded ethanols, and transferred to isoamylacetate, followed by criticalpoint-drying with liquid carbon dioxide. After coating with gold, specimens were observed under a scanning electron microscope (AMR Leitz 1200B) under an acceleration voltage of 15 kV . RESULTS
The MTT assay showed a significant increase in mitochondrial activity in cell populations that had been subjected to EMF for 14 days. The difference in mitochondrial activity between EMF-stimulated cells and control cells was clearly detectable (31%) when low numbers of cells (10 cells/well) had been seeded . Differences were less (17-21%) for initially higher numbers of cells (20-50 cells/well) . Starting with 100 cells/well the MTT reduction approached a plateau for both control and exposed cells (Fig . 2). Cells alternately exposed to EMF (3 days of intermittent EMF application followed by 3 days of culturing without EMF) showed a higher MTT reduction than control cells (without EMF), but a lower MTT reduction than cells with continuous 6 day EMF exposure . Regardless of the initial cell concentration there
130
0,10 100
200 500 initial cell count/ml
1,000
Fig . 2. Reduction of MTT in MCF7 cells incubated for 14 days and exposed to EMF (A) and omitting EMF (0) . Cell counts as seeded at the beginning of the experiment . Shown are the means of 36 measurements out of three independent experiments . * : p < 0.05 .
was a significant increase in MTT reduction in EMF-stimulated cells to unstimulated cells after 6 days exposure (Fig . 3) . SEM revealed morphological alteration of the cell surface due to EMF . Membrane ruffling and microvilli formation are more pronounced under EMF . Intercel-
0,0
100
500
1,000 2,Q00 initial cell count/well
5y000
Fig. 3. Determination of MTT reduction in MCF7 cells under the influence of EMF for 6 days (A), without the influence of EMF (0) and three days under EMF followed by three days without (x) . Shown are means of 60 measurements out of five independent experiments .
131
Fig. 4 . SEM image showing MCF7 control cells incubated for 72 h . Magnification 1920 X . lular junctions remained unaltered . The arrangement of villous projections was not affected by exposure to EMF (Figs. 4 and 5) . DISCUSSION AND CONCLUSION As shown by Mosmann [11] mitochondrial activity of the cell population correlates the number of cells . Therefore, at the end of each experiment mitochon-
Fig . 5 . SEM image showing MCF7 cells after exposure to EMF for 36 h (total incubation time 72 h) . Magnification 1920 X .
132
drial activities of the cell populations were determined by means of the MTT assay . Under the influence of EMF, MCF7 cells showed an increased MTT reduction compared with the control over a broad range of initial population densities . This effect was first detectable after 6 days of intermittent exposure and was even more pronounced after 14 days . This effect could first be detected after two days continuous exposure (data not shown) . Increased MTT reduction correlated to morphological changes in that the surface structure of MCF7 cells appeared more rough and uneven . EMF characteristics, culturing conditions, and the cell types used clearly demand further specifications as changes vary with different experimental settings . For example, Akamine et al . [121 reported on pulsed magnetic field treatment of embryonal carcinoma cells which showed regular arrangement and smoother surface then control cells. The positive effect of EMFs shown in clinical applications is also substantiated in the present paper . Nevertheless knowledge as to why EMFs may produce beneficial effects in vivo is still patchy . Future work should therefore be directed towards the understanding of the (molecular) mechanisms by which EMFs interact with cell replication and cell proliferation . REFERENCES 1 W . Kraus and F . Lechner, Munchner Med. Wochenschrift, 42 (1972) 1814 . 2 R. Ascherl, G . Blumel, F . Lechner, W. Kraus, Biochem . Bioenerg ., 14 (1985) 161 . 3 W. Mnhlbauer, Langenbecks Arch. Chir., 337 (1974) 637. 4 F . Tabrah, M . Hoffmeister, F . Gilbert, S . Batkin, C. Bassett, J. Bone Mineral Res., 5 (1990) 437. 5 E. Azavedo, Clin . Radiol., 43 (1991) 84 . 6 K . Takahashi, I . Kaneko, M . Date and E. Fukada, Experientia, 42 (1986) 185 . 7 J.C . Murray and R .W. Famdale, Biochim . Biophys . Acta, 838 (1985) 98 . 8 Y . Omoto, M . Hosokawa, M . Komatsumoto, T. Namieno, S . Nakajima, Y. Kubo and H . Kobayashi, Jpn. J. Cancer Res ., 81 (1990) 956 . 9 M. Petrini, L. Mattii, A. Sabbatini, G. Carulli, B. Grassi, R . Cadossi, G. Ronca and A. Conte, J. Bioelec., 9 (1990) 209 . 10 W. Kraus, Orthopade, 13 (1984) 78. 11 T. Mosmann, J. Immunol. Meth.,_65 (1983) 55 . 12 T . Akamine, H . Muramatsu, H. Hamada and T. Sakou, J. Cell. Physiol., 124 (1985) 247 .