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Doi:10.1016/j.bbrc.2005.08.243Biochemical and Biophysical Research Communications 336 (2005) 1144–1149 Induction of tamoxifen resistance in breast cancer cells Rainer Girgert a,*, Hartmut Schimming b, Wolfgang Ko¨rner c, Carsten Gru¨ndker a, a Department of Obstetrics and Gynecology, University of Go¨ttingen, D-37099 Go¨ttingen, Germany b Facility for Electronic Equipment, University of Ulm, D-89075 Ulm, Germany c Bavarian Environmental Protection Agency, D-86179 Augsburg, Germany The incidence of breast cancer in western societies has been rising ever since the Second World War. Besides the exposure to a mul- titude of new chemical compounds, electromagnetic ﬁeld exposure has been linked to breast cancer through a radiation-mediated anti-melatonin pathway. We investigated, whether low-frequency electromagnetic ﬁeld exposure interferes with the anti-estrogenic activity oftamoxifen. Two diﬀerent clones of the breast cancer cell line MCF-7 were exposed to highly homogeneous 50 Hz electromagnetic ﬁeldsand IC50 values were calculated from dose–response curves of tamoxifen at various ﬁeld intensities. An intensity-dependent shift oftamoxifen dose–response curves to higher concentrations with a maximal response at 1.2 lT was observed. Hypothetically, electromag-netic ﬁeld exposure could contribute to tamoxifen resistance observed in breast cancer after long-term treatment.
Ó 2005 Elsevier Inc. All rights reserved.
Keywords: Breast cancer; Estrogen receptor; Electromagnetic ﬁelds; Tamoxifen resistance; Dose–response The eﬀect of extremely low-frequency electromagnetic suﬀer of a high rate of mammary tumors if treated with the ﬁeld (ELF/EMF) exposure on human health has been widely debated. A number of epidemiological studies have (DMBA). Exposure of these rats to a 100 lT electromag- pointed to a slight increase in malignant diseases in popu- netic ﬁeld for 27 weeks increased the number of tumor lations exposed to electromagnetic ﬁelds through the vicin- bearing rats to 65% compared to 50% in sham exposed rats ity of power lines. A signiﬁcant positive association was . Although radiation energy of an extremely low-fre- observed between childhood leukemia and exposure of quency magnetic ﬁeld (50 Hz) is considered to be by far children to magnetic ﬁelds during the night In two stud- too low to induce DNA strand breaks, Lai and Singh ies, premenopausal women exposed to environmental ﬁelds observed an increase in DNA single- and double-strand stronger than 0.2 lT had an increased risk of breast cancer breaks in brain cells of rats exposed to electromagnetic (BC) Conversely, studies from Finland and Taiwan ﬁelds as low as 10 lT. This eﬀect was attributed to the gen- did not ﬁnd any increased BC risk in populations living eration of oxygen radicals in the presence of iron ions .
in the proximity (100–500 m) of power lines In addition, EMF was reported to suppress the nocturnal These epidemiological observations prompted the exam- synthesis of melatonin in the pineal gland in animals and ination of the impact of electromagnetic ﬁelds on breast human As melatonin may physiologically inhibit cancer incidence in an animal model. Sprague–Dawley rats estrogen production by the ovary, the EMF-suppressedmelatonin secretion would favor the growth of estrogen-de- pendent BC A direct oncostatic eﬀect of melatonin on Corresponding author. Fax: +49 5513912784.
E-mail address: (R. Girgert).
breast cancer cells was ﬁrst demonstrated by Blask and Hill 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.08.243 R. Girgert et al. / Biochemical and Biophysical Research Communications 336 (2005) 1144–1149 coiled around energized with an anti-parallel current, so that the net applied Inﬂuence of low-frequency magnetic ﬁelds on tumor cell gene expression static magnetic ﬁeld by the heating coil is annulled. On top of the heating coila second layer of copper wire is coiled around and connected to a signal generator delivering a 50 Hz sinusoidal alternating current. The current inducing ELF/EMF is regulated by electronic feedback stabilizing the chosen ﬁeld intensity. Feedback signals are generated by a Hall sensor measuring the ﬁeld intensity in the incubatorÕs center. Due to dimensions of the ﬁeld inducing coil, homogeneity of induced magnetic ﬁelds in a centralspace harboring the culture plates varies by less than ±5%. The temperatureinside the incubator is measured by a thermistor probe regulating the current and many other investigators thereafter. Melatonin re- to the biﬁlar heating coil. CO2 chamber concentration is kept at 5.0 ± 0.1% duced the growth of the estrogen receptor positive breast by an infrared sensor (Vaisala, Vanha, Finland) that regulates CO2 inﬂux cancer cell line MCF-7 in vitro by 18–27%. When the cells through a magnetic valve placed at a distance of more than 1 m outside the were exposed to a 60 Hz electromagnetic ﬁeld of 1.2 lT ﬂux Proliferation assay. Five hundred cells per well were plated into 96-well density, this inhibitory eﬀect of melatonin was completely plates (Falcon, Heidelberg) in 100 ll DMEM/5% fetal calf serum (FCS, blocked This surprising observation has been indepen- Biochrom, Berlin) without phenol red, 2 mM glutamine, 50 U/ml peni- dently replicated by several other authors Since the cillin/streptomycin, 2.5 lg/ml amphotericin B, and 1:100 non-essential oncostatic eﬀect of melatonin was estrogen-dependent, amino acids. After cell attachment, 100 ll medium or 100 ll tamoxifen Harland et al. tested whether the growth-inhibitory eﬀect solution at increasing ﬁnal concentrations of 10À8–5 · 10À6 M was addedto the wells in six replicates. Cells were exposed to magnetic ﬁeld inten- of the estrogen receptor (ER) antagonist tamoxifen was sities of 0, 0.2, 1.2, 10 or 100 lT, respectively, for seven days at 37 °C, 5% modulated by ELF/EMF exposure. Using the same exper- CO2. Cell number was determined by a colorimetric assay using Alamar imental set-up a reduced growth inhibition by tamoxifen Blue (Biosource, Solingen, Germany). The optical density (OD) of the on MCF-7 cells was observed at 1.2 lT These results reduced dye is assessed at 570 nm vs 630 nm after 4 h at 37 °C.
were also reproducible by other laboratories Calculation of dose–response curves. Means and standard deviations of the OD of six replicates were calculated. The proliferative eﬀect (PE) at The reduced tamoxifen activity in the presence of elec- each tamoxifen concentration was determined tromagnetic ﬁelds appears similar to a phenomenon, called‘‘tamoxifen resistance.’’ Tamoxifen has been used for treat- ment of ER positive BC for nearly thirty years. While most Dose–response curves for tamoxifen were obtained for each ﬁeld exposure patients with advanced estrogen-responsive BC initially condition by plotting the mean PE of all experiments versus the concentra- proﬁt from tamoxifen treatment, most of their tumors re- tion of tamoxifen on a half-logarithmic scale.
cur and respond no longer to tamoxifen treatment For calculating EC50 values of growth stimulation and the IC50 values Numerous investigations on EMF-regulated gene expres- for growth inhibition by tamoxifen, dose–response curves were split intotwo ranges, one, at lower concentrations (10À8–10À7 M) where tamoxifen sion in tumor cells yielded controversial results agonistically stimulated the growth of the MCF-7 cells, and the other The authors employed diﬀerent cellular systems and ranging from 10À7 to 5 · 10À6 M where tamoxifen inhibited the cell exposure conditions making comparisons between reported growth in an anti-estrogenic manner. Calculations of EC50 and IC50 were results diﬃcult. However, there is agreement in the necessi- performed using a VBA program for EXCEL 5 written by Josef Greve at ty of further investigations. In order to minimize uncon- the Fraunhofer Institute for Molecular Biology and Applied Ecology,Schmallenberg, Germany trolled external interferences with exposure conditions,particular caution must be paid to the generation of a sta-ble and reproducible magnetic ﬁeld.
For the analysis of EMF-induced modulation of tamox- ifen activity we developed and constructed a novel incuba- Inﬂuence of EMF on the anti-proliferative eﬀect of tor for the reproducible exposure of cells to deﬁned ELF/ EMF. Maximum eﬀort was employed to achieve highlyhomogeneous sinusoidal ﬁelds and control of exposure Dose–response curves of tamoxifen were calculated for two diﬀerent subclones (MCF-7 p40 and MCF-7 p181)and compared at various ﬁeld intensities.
The tamoxifen dose–response curves in either a shielded conﬁguration excluding surrounding environmental ﬁelds Cell culture. The human BC cell line MCF-7 was obtained from ATCC (0 lT), at the ambient (%0.2 lT) ﬁeld, and at sinusoidal (Manassas, USA). A second MCF-7 clone (MCF-7 p181) was provided by artiﬁcial ﬁelds of 1.2 and 100 lT intensity are shown in Dr. W. Ko¨rner, Augsburg. Cells were maintained in DulbeccoÕs modiﬁed . The results of the measurements at 10 lT are not MEM supplemented with 5% fetal calf serum (Biochrom, Berlin), 2 mMglutamine, 50 U/ml penicillin/streptomycin, 2.5 lg/ml amphotericin B, included in for a better clarity but the calculations and 1:100 non-essential amino acids (Biochrom, Berlin, Germany).
for IC50 of tamoxifen are listed in .
Exposure of cells to electromagnetic ﬁelds. We exposed MCF-7 cells to The dose–response curves of tamoxifen diﬀer clearly in various ﬁeld intensities (0, 0.2, 1.2, 10, and 100 lT) of a synthetic sinusoidal the two MCF-7 subclones examined (). The dose–re- 50 Hz alternating electromagnetic ﬁeld. Exposure- incubators with sinu- sponse curves of clone MCF-7 p40 (A) show an soidal current generator/regulators and separated CO2 blenders consistedeach of a copper tube, 30 cm in diameter and 75 cm in length, closed at either inhibitory eﬀect of tamoxifen on the growth of the BC cells end by heat accumulating copper plates. For heating, a biﬁlar copper wire is at concentrations >10À7 M. In the absence of any alternat- R. Girgert et al. / Biochemical and Biophysical Research Communications 336 (2005) 1144–1149 would be exposed to an average ambient magnetic ﬂux den-sity of %0.2 lT present in the laboratory. The dose–re-sponse curve of tamoxifen at 0.2 lT resembled the one recorded under shielded conditions. At 1.2 lT the dose–re-sponse curve is slightly shifted to the right, resulting in an IC50 value of 2.3 · 10À6 M. At a substantially higher ﬁeld shielded
intensity of 100 lT this shift of the dose–response curve is no longer observed and the IC50 of tamoxifen is reducedto about 0.9 · 10À6 M ( In the cell clone MCF-7 p181, the described eﬀects of magnetic ﬁelds on the dose–response curves of tamoxifen In the shielded situation (0 lT), the dose–response curve of tamoxifen in MCF-7 p181 cells showed a similar sigmoi- dal pattern as the one seen with the p40 clone. Even weak ambient ﬂux densities (0.2 lT) resulted already in a markedproliferative activity of tamoxifen at concentrations The maximal proliferative gain in MCF-7-p181 cells at 0.2 lT and a tamoxifen concentration of 10À7 M was 26% compared to the absence of tamoxifen.
Already at 0.2 lT the dose–response curve of tamoxifen was clearly shifted to higher concentrations. This shift waseven more pronounced at a ﬁeld intensity of 1.2 lT. The maximal proliferative eﬀect of tamoxifen at 1.2 lT was ob- shielded
served at a concentration close to 1 lM. At higher ﬁeld intensities (10 and 100 lT) the shift of the dose–response curve was lower as compared to 1.2 lT, but did not return to the values measured in the absence of the EMF These measurements clearly show a ‘‘window eﬀect’’ of the applied EMF with a maximum between 1.2 and10 lT as has also been observed in other biological systems Fig. 1. Dose–response curves of tamoxifen at various intensities of 50 Hz electromagnetic ﬁelds. (A) Clone MCF-7 p40. (B) Clone MCF-7 p181.
Cells were grown at increasing concentrations of tamoxifen either in ashielded conﬁguration (0 lT) (closed circle) or at 0.2 lT (open square) or Calculation of IC50- and EC50 values of tamoxifen at at 1.2 lT (upright triangle) or 100 lT (diamond). Cell number was estimated after 7 days of culture by a colorimetric assay. Control: cellnumber achieved in the absence of tamoxifen = 100%. Means of at least The dose–response curves of tamoxifen in MCF-7 three independent experiments with six replicates at each concentration.
(p181) cells (were separated into a proliferativebranch (10À8–10À7 M) and an anti-proliferative branch ing EMF (ambient ﬁeld shielded by a container of mu-met- (10À7–5 · 10À6 M) and EC50 values of the proliferative ef- al), the IC50 of tamoxifen was calculated at 1.4 · 10À6 M. If fect at low tamoxifen concentrations (clone p181 only) this mu-metal shielding were omitted, cells in culture plates and the IC50 values of the anti-proliferative eﬀect of tamox- Table 2IC50- and EC50 values of tamoxifen at various ﬁeld intensities IC50: tamoxifen concentration for half-maximal growth inhibition.
EC50: tamoxifen concentration for half-maximal growth stimulation.
R. Girgert et al. / Biochemical and Biophysical Research Communications 336 (2005) 1144–1149 ifen at high concentrations were calculated from the sepa- selection process favoring cells in the tumor that are al- rate dose response curves for all applied ﬁeld intensities ready sensitized to growth stimulation by tamoxifen or are at least insensitive to the growth inhibition or to cellu- In the shielded conﬁguration, clone p181 was double lar alterations induced by the drug or other environmental as sensitive to the inhibitory eﬀect of tamoxifen as clone factors. Wiseman et al. observed a sensitization of p40 If p181 cells were exposed to the ambient tumor cells to the proliferative activity of IGF-I after treat- EMF of about 0.2 lT, a threefold higher tamoxifen con- ment with tamoxifen. Tamoxifen treatment would select centration was needed to achieve 50% growth inhibition for these IGF-1-dependent cells ultimately producing a as compared to the shielded situation. In cells of clone p40, sensitivity to tamoxifen was only slightly reduced The modulated tamoxifen eﬀects that we observed in at 0.2 lT. A strong shift in the IC50 occurred in both cell clones at 1.2 lT and similarly high concentrations of incompatible with a selection process because the time tamoxifen were needed for a half-maximal inhibition at a of exposure was too short to allow a hypothetically magnetic ﬁeld of 10 lT. Surprisingly, at 100 lT the eﬀect tamoxifen-stimulated or at least tam-insensitive subpopu- on tamoxifen inhibition was clearly lower than at 10 lT.
lation to overgrow the majority of tamoxifen-sensitive From the data in it can be seen that ELF/EMF clearly reduce the growth-inhibitory eﬀect of tamoxifen One further hypothesis for the development of tamoxi- with a maximum eﬃcacy between 1.2 and 10 lT, and that fen resistance in breast tumors suggests that this resistance this eﬀect is waning at higher ﬁeld intensities.
is associated with an inappropriate expression of receptor A marked estrogen-like proliferative eﬀect at low tamox- interacting proteins (RIPs) A multitude of receptor ifen concentrations was only observed in clone p181 in the interacting proteins (RIPs) regulate gene transcription by presence of EMF. The proliferative EC50 is reduced with nuclear hormone receptors, e.g., ER, for review, see increasing ﬁeld intensities, reaching its strongest eﬀect at In a preliminary clinical study, high levels of SRC-1 were detected in breast tumors showing good response to tamox-ifen treatment In a comparison of the expression of various RIPs in wild type MCF-7 breast cancer cells and MCF-7/TAMR- Here we show that the anti-estrogenic activity of tamox- 1 cells that acquired a tamoxifen resistant phenotype ifen is reduced in two subclones of MCF-7 cells under the after permanent treatment with tamoxifen revealed no inﬂuence of ELF/EMF to diﬀerent extent. Dose–response diﬀerences in the expression of TIF-1, SUG-1, and curves of the growth-inhibitory eﬀect of tamoxifen are SMRT but RIP140 expression was lower in non-stimu- shifted towards higher concentrations leading to a reduced lated cells of the resistant strain. Stimulation of the growth inhibition at a given concentration. Our observa- resistant cells by E2 or tamoxifen increased the level tion conﬁrms results from a previous report describing a re- of RIP140 mRNA but not in the parental MCF-7 cells duced inhibitory eﬀect of tamoxifen at 10À7 M from 40% to only 17% by exposure to an EMF of 1.2 lT . More rel- When expression levels of the corepressor N-CoR are evant from a therapeutic point of view, in our experiments low, patients receiving tamoxifen therapy experience poor tamoxifen even enhanced growth of the MCF-7 cells at outcomes. This observation suggests that tamoxifen antag- concentrations below 10À6 M if cells were exposed to onism requires high levels of N-CoR function .
EMF. The behavior of breast cancer cells exposed to Tamoxifen can act as an agonist through ERa/ERb het- EMF appears similar to the frequently observed tamoxifen erodimers, thus, in breast cancer cells where suﬃcient con- resistance in tamoxifen-treated patients.
centrations of ERa and ERb are present, tamoxifen could About 40% of ER-positive breast tumors fail to respond induce cell proliferation An imbalance of ERa- and to anti-estrogen therapy by tamoxifen from the beginning ERb-expression may determine a breast tumor to become (intrinsic resistance), while most of the residual tumors that initially respond to tamoxifen develop resistant relapse in Exposure to ELF/EMF is omnipresent in our electriﬁed the course of treatment (acquired resistance—AR) environment but the strength of the EMF generated by the Tamoxifen is known as a partial estrogen antagonist be- electric wiring in usual households varies between 0.01 and cause it can either stimulate or inhibit ER-dependent 1 lT, in occupational situations exposure values of 1 lT tumor growth in a tissue-, cell-, and promoter-speciﬁc man- and more are occasionally achieved At 1.2 lT the ner. Like other selective estrogenic response modiﬁers enhancing/augmenting inﬂuence of ELF/EMF on the pro- (SERMs) tamoxifen acts estrogen antagonistic in certain liferative eﬀect of tamoxifen is strongest and is surprisingly tissues, e.g., breast tissue, and agonistic in other tissues like waning at higher ﬁeld intensities. Such kind of ‘‘window ef- bone and uterus . Resistant tumors behave like tissues fect’’ of EMF activity has also been observed in other where tamoxifen acts as an estrogen agonist.
Several mechanisms have been hypothesized as to how In the clinical situation where BC is frequently treated AR to tamoxifen could arise. AR may be due either to a with tamoxifen, it could be speculated that EMF exposure R. Girgert et al. / Biochemical and Biophysical Research Communications 336 (2005) 1144–1149 may also contribute to the induction of a tamoxifen-resis-  R.P. Liburdy, T.R. Sloma, R. Sokolic, P. Yaswen, ELF magnetic tance-like behavior in some breast tumors.
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