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      Inhibition of Cancer Cell Growth by Exposure to a Specific Time-Varying Electromagnetic Field Involves T-Type Calcium Channels

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          Abstract

          Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca 2+ influx which could be blocked by inhibitors of voltage-gated T-type Ca 2+ channels. Blocking Ca 2+ uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca 2+ influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.

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          Most cited references49

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          Cyclin A in cell cycle control and cancer.

          Cyclin A is particularly interesting among the cyclin family because it can activate two different cyclin-dependent kinases (CDKs) and functions in both S phase and mitosis. An embryonic form of cyclin A that is only essential for spermatogenesis is also present in some organisms. In S phase, phosphorylation of components of the DNA replication machinery such as CDC6 by cyclin A-CDK is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. In mitosis, the precise role of cyclin A is still obscure, but it may contribute to the control of cyclin B stability. Cyclin A starts to accumulate during S phase and is abruptly destroyed before metaphase. The synthesis of cyclin A is mainly controlled at the transcription level, involving E2F and other transcription factors. Removal of cyclin A is carried out by ubiquitin-mediated proteolysis, but whether the same anaphase-promoting complex/cyclosome targeting subunits are used as for cyclin B is debatable. Consistent with its role as a key cell cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.
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            Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects

            The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca2+/calmodulin stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein kinase G pathway stimulation. A well-studied example of such an apparent therapeutic response, EMF stimulation of bone growth, appears to work along this pathway. However, pathophysiological responses to EMFs may be as a result of nitric oxide-peroxynitrite-oxidative stress pathway of action. A single such well-documented example, EMF induction of DNA single-strand breaks in cells, as measured by alkaline comet assays, is reviewed here. Such single-strand breaks are known to be produced through the action of this pathway. Data on the mechanism of EMF induction of such breaks are limited; what data are available support this proposed mechanism. Other Ca2+-mediated regulatory changes, independent of nitric oxide, may also have roles. This article reviews, then, a substantially supported set of targets, VGCCs, whose stimulation produces non-thermal EMF responses by humans/higher animals with downstream effects involving Ca2+/calmodulin-dependent nitric oxide increases, which may explain therapeutic and pathophysiological effects.
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              Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.

              We have developed fluo-4, a new fluorescent dye for quantifying cellular Ca2+ concentrations in the 100 nM to 1 microM range. Fluo-4 is similar in structure and spectral properties to the widely used fluorescent Ca(2+)-indicator dye, fluo-3, but it has certain advantages over fluo-3. Due to its greater absorption near 488 nm, fluo-4 offers substantially brighter fluorescence emission when used with excitation by argon-ion laser or other sources in conjunction with the standard fluorescein filter set. In vitro, fluo-4 exhibited high fluorescence emission, a high rate of cell permeation, and a large dynamic range for reporting [Ca2+] around a Kd(Ca2+) of 345 nM. We have also developed several Ca(2+)-indicators related to fluo-4 having lower affinities for Ca2+ that are useful in cellular studies requiring quantification of higher [Ca2+]. In a variety of physiological studies of live cells, fluo-4 labeled cells more brightly than did fluo-3, when challenged with procedures designed to elevate calcium levels. Fluo-4 is well suited for photometric and imaging applications that make use of confocal laser scanning microscopy, flow cytometry, or spectrofluorometry, or in fluorometric high-throughput microplate screening assays. Because of its higher fluorescence emission intensity, fluo-4 can be used at lower intracellular concentrations, making its use a less invasive practice.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                14 April 2015
                2015
                : 10
                : 4
                : e0124136
                Affiliations
                [1 ]Departments of Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada
                [2 ]Regional Cancer Program, Health Sciences North, Sudbury, Ontario, Canada
                [3 ]Behavioural Neurosciences, Laurentian University, Sudbury, Ontario, Canada
                [4 ]Northern Ontario School of Medicine, Sudbury, Ontario, Canada
                Dalhousie University, CANADA
                Author notes

                Competing Interests: Co‐author Robert Lafrenie is a PLOS One Editorial Board member, but this has not altered the authors' adherence to the PLOS ONE editorial policies and criteria.

                Conceived and designed the experiments: CAB SAK MAP RML. Performed the experiments: CAB ALB RML. Analyzed the data: CAB ALB RML. Contributed reagents/materials/analysis tools: SAK MAP RML. Wrote the paper: CAB ALB SAK MAP RML.

                Article
                PONE-D-14-30053
                10.1371/journal.pone.0124136
                4397079
                25875081
                84295f8a-2b8c-4e84-8dec-e83e01c3c88a
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 4 July 2014
                : 10 March 2015
                Page count
                Figures: 7, Tables: 0, Pages: 16
                Funding
                This work was supported by the Northern Cancer Foundation, Sudbury, Ontario and Natural Sciences and Engineering Research Council, Canada (MAP, primary). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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