Abstract Objective To investigate the inhibitory effect of extremely low frequency electromagnetic fields on cultured tumor cells. Human osteosarcoma cells (MG63), breast cancer cells (MCF7) and hepatoma cells (HepG2) was inoculated into 96-well plates, and placed in the magnetic field strength of 10mT, 20mT, 40mT, 60mT’s magnetic field intervention, stimulation 2 times a day, every 1h, for 3 days, while the control group in each cell is set under normal conditions. 3 days after the MTT assay cell activity (OD) and detected in three cell apoptosis by flow cytometry (Annexin V-FITC and PI double standard). Results after 72h detected by MTT OD value of the three kinds of cells, and comparison to the normal control group, the SPSS software statistical results showed that the OD value of the magnetic field group than the normal group (P <0.05); flow cytometry results show positive magnetic field The apoptotic cells is higher than the normal group, the difference between the two groups was considered statistically significant. Conclusion extremely low frequency electromagnetic fields significantly inhibited tumor cells induced apoptosis of tumor cells to a certain extent.

【Key Words】 extremely low frequency electromagnetic fields of tumor cell apoptosis MTT

   Now many domestic and foreign scholars have applications in magnetic field treatment of tumor coverage, and achieved a certain effect. Relatively small magnetic field away from the body of tumor cells and its principle reported, this study uses extremely low frequency electromagnetic fields intervention logarithmic growth phase three kinds of tumor cells and tumor cells under normal growth conditions compared aims understanding of the magnetic field on the in vitro growth of tumor cell proliferation, differentiation, provided the experimental basis for the magnetic field treatment of cancer.

    1 Materials and methods

    1.1 Materials

    1.1.1 The source of the magnetic field of very low frequency electromagnetic fields provided by the the Wuhan Naval Engineering College (Model: DL-D03 Tianjin Central Electric Co., Ltd.). Rated voltage 250V, single-phase frequency of 50Hz, adjustable transformer and resistor adjusts the output magnetic field strength.

    1.1.2 The object of study of three tumor cells HepG2 by Tongji Medical College, Huazhong University of Science and Technology Experiment Center of Liver Surgery, Tongji Hospital, to provide, MG63 and MCF7 saved by the laboratory.

    1.1.3 Main reagents fetal bovine serum and 1640 medium Hyclone Company. MTT, dimethyl sulfoxide (DMSO), Annexin-V / FITC and PI, carbon dioxide incubator, inverted microscope, fluorescent microscope, ELX  the 800UV type ELISA tester, flow cytometry (Department of Immunology, Tongji Medical College).

    1.2 Experimental Methods

    1.2.1 tumor cell growth morphology of intervention before and after the intervention 72h, three kinds of cells in each group growth morphology was observed under phase contrast microscope.

    1.2.2 MTT (methyl thiazolyl tetrazolium blue) colorimetric determination of cell viability to the third generation of the three kinds of cells cultured with 0.25% trypsin digestion, wind and percussion made from a single cell suspension (after adjusting the cell density of 1.0 × 104), were inoculated with 5-96 orifice. Each block 96-well plates 1 to 3 as a cell-free serum, the serum substance in order to eliminate the influence of the light absorption value; 4 to 6 as MG63 cell suspension; 7 to 9 as a MCF7 cell suspension; 10 ~ 12 classified suspension of HepG2 cells. A 96-well plate for the normal control group, placed at 37 ° C, 5% CO2 incubator; 2 to 5 plates were placed in 37 ° C, 5% CO2 for incubator containing magnetic field coil. 96 well plate is put in the center of the coil to reach a preset magnetic field strength by adjusting the transformer. 2 to 5, the magnetic field intensity of the 96-well plate were 10mT, 20mT, 40MT, 60mT, stimulation time 2 times a day, each time for 1H. 3 days after the MTT solution was added to each well 20μl, incubated for 4h after termination of culture. Carefully aspirate the supernatant was discarded, and each orifice Add DMSO 150μl melting crystalline material, oscillation 10min after the absorbance value of each well was measured on a microplate reader.

    1.2.3 Flow cytometry time of the magnetic field strength and the magnetic field detected by the proportion of each group of three kinds of tumor cell apoptosis and the same way as before. Magnetic stimulation stopped, trypsinized cells and adjust cell concentration of 1 × 106 cells / ml. 1000r/min 4 ℃ 1ml of cell centrifugal 10min, the supernatant was discarded; adding 1ml 4 ℃ PBS gently shaken to the cell suspension, 4 ° C 1000r/min centrifugal 10min and the supernatant was removed, a total of three times to repeat the steps; cells suspended 250μl of binding buffer, taking in 100 μl of cell suspension in 5ml flow tube, add 5μl Annexin-Ⅴ / FITC and 10μl PI solution, mix and incubated in the dark at room temperature for 15min, 400μl of PBS was added in the reaction tube After flow cytometry (FACS) detection analysis. 5 samples in each group, and the count of 1 × 106 cells per sample, and calculate the positive rate. At 480nm excitation light, to detect the proportion of apoptotic cells in total cell numbers in the three kinds of tumor cells.

    1.3 Statistical

    The experimental results ± s, using SPSS 11.0 statistical software, and the application of single-factor analysis of variance was used for statistical.

    2 Results

    2.1 tumor cell growth morphology observed

    After the intervention to 72h, significantly the growth of the magnetic field in each group and the control group of tumor cells and normal cells full volume, cell density; magnetic field group cell volume than normal group of small, sparse density, adherent cells shedding apoptotic cells floating increased significantly (collect three kinds of growth of adherent cells magnetic field through the pre-experimental intervention after 72h cell suspension flow analysis).

    2.2 MTT (methyl thiazolyl tetrazolium blue) colorimetric determination of cell viability

    The mean optical density value of each group in different strength magnetic field stimulation (OD value, ± s) (see Table 1) and the magnetic field intensity on the tumor inhibition rate (Table 2). The results can be seen in the normal control group, the three kinds of tumor cell growth state, its OD values ​​were higher than the magnetic field group; OD value of the magnetic field group have different degrees of decline, its maximum inhibition rate of 34.82% (of MG63 , 20mT), MG63 and MCF7 20mT maximum inhibition rate (34.82%, 31.06%), respectively, the HepG2 in 60mT maximum inhibition rate (19.69%). 0.05)。">By the SPSS software variance analysis, the difference between the magnetic field with the control group was significant (P <0.05), the difference was not statistically significant (P> 0.05) in the magnetic field strength between groups. Table 1 three tumor cell OD value Note: Compared with the normal group, * mark: P <0.05 Table 2 magnetic field on tumor cell growth inhibition rate

    2.3 Flow cytometry results

    This experiment was based on the results of MTT, flow cytometry, i.e. in the three kinds of tumor cells in the MTT assay on a magnetic field under the conditions of the maximum inhibition rate of flow cytometry analysis. There are other factors, to take five sample tubes each condition, and set the comparison group under normal conditions, as shown in Figure 1 to 4, such as the apoptotic rate of the normal control group in the same conditions, greater than 5%, then consider affect the results of this experiment, the sample tube to give up the statistics. As a result, data ± s, as shown in Table 3. Table 3 different magnetic field intensity on the inhibition of tumor cells

    3 Discussion

    Many scholars have studied the magnetic field immunity of tumor-bearing animals and in vitro tumor cell biological traits affect [1,2]. Field type and intensity of use, the biological effects are not the same. And, even in the same magnetic field conditions, a variety of tumor cells can produce different results, we found in preliminary experiments in the the 50Hz alternating magnetic field of in vitro cultured tumor cells inhibited [3]. To further investigate the role of the magnetic field on a variety of tumor cells, we chose three kinds of tumor cells in a variety of magnetic field strength, and analysis of their results as follows:

    You can see from our experiments, the magnetic field for a variety of tumor cells were significantly inhibited. Santi et al [4] are also in the experiments prove that the magnetic field can inhibit the growth of tumor cells, and can significantly extend the life of the nude, and in the course of the experiment, we further observed that the inhibitory effect of magnetic field on tumor cells is mainly induced apoptosis of tumor cells, rather than killing effect. If the cycle of the culture over a certain time period (eg, 10 days) in the training process, the three kinds of tumor cells and normal tumor cells in the four kinds of magnetic field strength under the action of the MTT detection and no obvious flow cytometry 0.05)。">difference (P> 0.05). Once the tumor cell growth rate over the inhibitory effect of the magnetic field, the excessively long culture results will be the tumor cells covered the entire culture bottle. We chose the logarithmic growth phase cells using MTT and flow cytometry to detect apoptosis of tumor cells.

    The magnetic field on the induction of apoptosis of tumor cells is not the strength of the magnetic field was a simple linearly proportional relationship. Was observed in the experiment, the experiment is too small when the strength of the magnetic field (magnetic field strength <1mT), three kinds of tumor cells in the MTT assay and flow cytometry, and no significant differences; while the strength of the magnetic field is too large, the magnetic field will also affect the normal cell division process, its constituting a direct injury [5], this too strong magnetic field strength, and its feasibility questionable treatment of tumor effect, our experiments can be seen that different cell in a certain kind of magnetic field strength induced apoptosis, which MG63 and MCF7 are in the 20mT maximum inhibition rate (34.82%, 31.06%), while the maximum inhibition rate (19.69%) in 60mT HepG2. We only discussed the reference values ​​of the three kinds of tumor cells, and a variety of tumor cell apoptosis induction magnetic field strength most suitable for worthy of further investigation. Huo Xiaolin et al [6] reported HepG2 cells by 0.2T static magnetic field dealt with separately 15min, 30min and 24h after MTT test results showed no significant difference between the experimental and control groups. The difference between the results of these experiments further indicate that the mechanism of the interaction between the magnetic field and the cells are quite complex, the different magnetic field strength, the magnetic field type, a different exposure magnetically, and the different cell types, are interested in the experimental results will produce different effects [7], which is further evidence that the role of the magnetic field on the tumor cells is not only by certain molecules, and some single mechanism to achieve.

    The role of the magnetic field induces tumor cell apoptosis may be related to the following mechanisms: (1) tumor cell growth and division strong in mitosis (M phase) and DNA synthesis phase (S phase) cells more, relative to normal cells, more sensitive; its role on the magnetic field (2) under the effect of magnetic field, the trajectory of the charged particles within the tumor cells susceptible to interference, the molecules within the cell and a little role and delivery of the particles susceptible to interference, affecting the entire physiological function of cells , so that the growth of tumor cells and reproduction affected by [8]. Extracellular Ca2 + concentration higher intracellular Ca2 + concentration of about 1000 times. Mitochondrial Ca2 + concentration than the cytoplasm Ca2 + concentrations 1000 times. Pulsed magnetic field generated by transmembrane potential changes will affect the extracellular Ca2 + influx and cause mitochondrial Ca2 + outflow. Ca2 + concentration of intracellular signal system signal, the transmembrane potential change is bound to change the state of membrane ion channels, leading to changes in the ion concentration [9]; (3) magnetic field can cause charged charged groups macromolecules within the tumor cells ( such as: the conformational change of the enzyme), which affects the play of their biological activity [5]. (4) The magnetic field acts on the receptor on the cell membrane and the cell membrane, the cell signal transduction networks mediated effect, the signal transmitted to the cells, causing a corresponding biological effects [10]. Electromagnetic fields – film and membrane receptors – transmembrane signal transduction – the biological effects of further affect the biological behavior of the tumor cells.

    View from home and abroad, although the mechanism is more complex magnetic field on tumor effects, some of these factors there are uncertainties, but the the application magnetic field as a treatment of tumors, has in many ways made a certain effect, although this kinds of effects are mostly to relieve symptoms and prolong life time, but it is also meaningful, we need to further explore.