Research ArticleExperimental Studies

Static Magnetic Stimulation Induces Cell-type Specific Alterations in the Viability of SH-SY5Y Neuroblastoma Cell Line

HELOUISE R. MEDEIROS, JOSÉ A. F. ASSUMPCAO, LICIANE F. MEDEIROS, MARTINA STAPENHORST, LARA NUNES, NICOLE A.C. HENCKES, CAROLINA URIBE CRUZ, FELIPE FREGNI, PAULO R.S. SANCHES, FERNANDA S.O. OLIVEIRA, WOLNEI CAUMO, ELIZABETH O. CIRNE-LIMA and IRACI L.S. TORRES
Anticancer Research September 2020, 40 (9) 5151-5158; DOI: https://doi.org/10.21873/anticanres.14518

Abstract

Background/Aim: Magnetic stimulation is used in the treatment of a diversity of diseases, but a complete understanding of the underlying mechanisms of action requires further investigation. We examined the effect of static magnetic stimulation (SMS) in different cell lines. Materials and Methods: A culture plate holder with attached NeFeB magnets was developed. Different magnetic field intensities and periods were tested in tumoral and non-tumoral cell lines. To verify the cellular responses to SMS, cell viability, cell death, cell cycle and BDNF expression were evaluated. Results: Exposure of SH-SY5Y cells to SMS for 24 hours led to a decrease in cell viability. Analysis 24 h after stimulation revealed a decrease in apoptotic and double-positive cells, associated with an increase in the number of necrotic cells. Conclusion: The effects of SMS on cell viability are cell type-specific, inducing a decrease in cell viability in SH-SY5Y cells. This suggests that SMS may be a potential tool in the treatment of neuronal tumors.

Over the years, several electrophysiological studies have expanded the understanding of normal brain activity and its pathological conditions. Technological advances have been an important part of the improvement of therapies and research in several areas such as neurology, psychology, and psychiatry (1). Brain stimulation is a tool for modulating brain function, allowing the association of activity patterns and cognitive function to establish cause-consequence relations (2). Brain stimulation techniques are usually divided into two different types: invasive and non-invasive (NIBS) techniques. Whereas invasive techniques involve greater risk for patients, as demonstrated by studies that compare the impact of different procedures (3, 4), non-invasive techniques have shown favorable results together with lower risks (3, 5). Indeed, NIBS's application has been described in different scenarios: 1. Cognitive improvement on depression (6, 7); 2. Improvement in post-stroke recovery (8); 3. Improvement of the memory and the quality of life of patients with Alzheimer's disease (9, 10); 4. Relief of chronic pain (11, 12). These findings, alongside a reduction in induced adverse effects, set NIBS as promising alternatives for the treatment of several diseases and neurological disorders (13).

Recently, in vitro studies reporting stimulation using non-invasive techniques have been reported. Potential uses and effects of magnetic stimulation over cellular processes have been described, particularly using static magnetic stimulation (SMS). Apart from physiological and homeostatic events, such as wound healing (14), SMS has shown effects in cancer models of glioblastoma (15), adenocarcinoma (16) and leukemia (17), where it has been shown to control the cell cycle, reduce drug resistance to cisplatin and enhance natural killer cell cytotoxicity against tumor cells, respectively.

SMS, unlike repetitive transcranial magnetic stimulation (rTMS) – in which changes in the magnetic field create an electric current through electromagnetic induction - does not induce electric currents; however, it has been shown to influence a variety of biological systems (18). Transcranial stimulation with a static magnetic field applied in humans reduces the excitability of the motor cortex for a few minutes after the end of the stimulation (19). Few studies have explained the effects of SMS on nervous cells. A comparison between renal cells and cortical astrocytes in rats showed that SMS decreases proliferation and increases apoptosis and necrosis in renal treated cells, while the opposite effect was seen in cortical astrocytes; stimulated cells showed more proliferation and less cell death (20). These results suggest that different cell types can respond differently to SMS.

Immortalized cell lines are widely used models for in vitro studies, for their ease of maintenance, high proliferative rates, highly homogenous and reproducible results (21). In this context, the human neuroblastoma cell line SH-SY5Y is often used for neuronal cell studies, since SH-SY5Y cells can be differentiated in dopaminergic neurons (22).

This study aimed to establish a method for in vitro SMS, to investigate its effects on cell viability, cell death and the cell cycle of different cell lines, and determine whether the responses are cell type specific.

Materials and Methods

Cell culture and differentiation. Adipose-derived mesenchymal stem cells, human vaginal malignant melanoma HMVII cell line and human neuroblastoma SH-SY5Y cell line, were obtained from the Banco de Células do Estado do Rio de Janeiro (Rio de Janeiro, Brazil). Mesenchymal stem cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% heat-inactivated Fetal Bovine Serum (FBS) (GIBCO) and 1% Penicillin/Streptomycin (GIBCO) at 37°C and 5% CO2. HMVII cells were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640 (GIBCO) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (GIBCO) at 37°C and 5% CO2. SH-SY5Y cells were maintained in 1:1 Ham's F12 and DMEM Low Glucose (GIBCO) supplemented with 10% heat-inactivated FBS (GIBCO), and 1% penicillin/streptomycin (GIBCO) at 37°C and 5% CO2. Cells were passaged at 80-90% confluency. Cells were seeded in 24-well plates (using only 6 wells per plate, according to Figure 1) at a density of 1×106 cells per well and kept at 37°C and 5% CO2. Differentiation was induced 24 h after plating using 1:1 Ham's F12 and DMEM Low Glucose (GIBCO) supplemented with 1% heat-inactivated FBS (GIBCO), 1% penicillin/streptomycin (GIBCO) and 10 μM Retinoic Acid (RA). The RA-containing culture medium was replaced every three days until day 10. Evaluation of cell morphology and differentiation was done using phase-contrast light microscopy.

Static magnetic stimulation (SMS). Stimulation with SMS was done using a specially designed stand for attachment of standard 24 well plates. Each stand contains six NdFeB (neodymium-iron-boron) magnets with cylindrical shape (12 mm diameter and 6 mm height), spaced out so that the magnetic fields do not interact (Figures 1 and 2). The distribution of the magnets is made so that they are coupled exactly to 6 wells of a 24-well plate. Each 24-well plate, therefore, is seeded in only 6 wells. There is an adjustment for the distance between the culture plate and the stand to guarantee the strength of the magnetic field. The magnetic field traversed a layer or several layers of cells, in the same way, i.e., cell grouping, or density does not change the field's intensity. The adjustment of the magnetic field was performed with a customized screw, and the measurement of the magnetic field was done using a Hall Effect Gaussmeter (Wuntronic GmbH, Germany), available at the HCPA Biomedical Engineering Laboratory. Stimulation for the initial MTT assay using only the SH-SY5Y cell line was done using intensities of 0.1 T, 0.2 T and 0.3 T (±2% tolerance), for 60 min. The remaining stimulations were performed for 24 h from plating, with an intensity of 0.3 T. Control groups did not receive stimulation. The 48-h group received stimulation during the first 24 h only.

MTT assay. The MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, Brazil) is a colorimetric assay that reflects cell viability. Immediately and 24 h after SMS exposure, cells were incubated with MTT in saline (132 mM NaCl, 4 mM KCI, 1 mM CaCl2, 6 mM glucose, 10 mM HEPES, pH 7.4). Without removing the medium from the cells, 0.75 mg/ml MTT was added, incubated for 1 h at 37°C and dimethyl sulfoxide (DMSO) was added for cell disruption. The absorbance was determined at a wavelength of 570 nm, using a wavelength of 620 nm as a reference in a spectrophotometer. Cell viability was expressed as a percentage relative to the absorbance determined in the control cells.

Cell death (PI/Hoechst staining). Viable and dying cells were identified after staining of the nuclei with Propidium Iodide (PI) (Thermo Fischer, Carlsbad, CA, USA) and Hoechst 33342 (HO) (Sigma Aldrich, Willow Creek Road, Eugene, EUA). The cells were incubated in a solution containing PI and HO 5 mg/ml for 15 min and visualized by fluorescence microscopy. To quantify the number of dead/alive cells, ten photos per well were taken, randomly chosen. Images were analyzed using ImageJ software.

Cell death (Annexin-V/PI staining). Annexin-V/PI staining was performed to obtain a more detailed profiling of SH-SY5Y cell death. Apoptotic cells lose the asymmetric disposition of membrane components and proteins, such as phosphatidylserine residues, usually found on the inside sheet of the plasma membrane. Upon entering apoptosis, these proteins are exposed to the outer sheet of the plasma membrane, where it is made available for Annexin-V-FITC staining. On the other hand, necrotic cells lose membrane integrity and are, therefore, positively stained using PI. In this manner, the different events surrounding cell death can be distinguished from one another according to different staining profiles. Double-positive cells do not have a clear phenotype. These cells can be either late-stage apoptotic or necrotic cells. Apoptotic cells (Annexin-V+/PI−), necrotic cells (Annexin-V−/PI+), and alive cells (Annexin-V−/PI−) are quantified through flow cytometry. After treatments, the samples were washed with PBS, resuspended in 100 μl of Annexin-V Binding Buffer 1X and incubated with 2.5 μl of Annexin-V FITC for 15 min, at room temperature, protected from light. The samples were incubated in Propidium Iodide solution (2 μg/ml), an additional fluorescent marker, in Annexin-V Binding Buffer 1X for 5 min at 4°C protected from light. Alive cells show membrane integrity, which prevents PI from entering the cell and staining nucleic acids (RNA and/or DNA). The samples were immediately analyzed by flow cytometry using the Attune® Acoustic Focusing Cytometer (Applied Biosystem- Life-Thermo). As an experimental control, apoptosis was induced using 20% DMSO for 15 min and necrotic cells were obtained by heating the cells at 70°C for 15 min.

Figure 1.
Figure 1.

Schematic for the static magnetic stimulation device.

Cell cycle. Treated cells were resuspended in 100 μl of PBS and 900 μl of ice-cold 70% ethanol were added and incubated for 1 h at 4°C. Samples were centrifuged at 5000 rpm for 10 min and the pellet was washed three times in PBS 1X and resuspended in a standard staining solution (0.1% Triton X-100, 100 μg/ml PI and 50 μg/ml DNAse-free RNAse) for 15 min at 37°C, protected from light. Samples were resuspended in PBS for immediate flow cytometry analysis using the Attune® Acoustic Focusing Cytometer (Applied Biosystem-Life-Thermo).

BDNF expression. Total RNA was extracted as recommended by the manufacturer using the RNeasy Mini Kit (Qiagen, Austin, TX, USA). Complementary DNA was synthesized from 1 μg RNA using SuperScriptVILOtm (Invitrogen, Brazil). PCR reactions were prepared using MasterMix TaqMan (Applied Biosystems, Germantown, MD, USA) and StepOne™ Real-Time PCR System (Applied Biosystems). Real-time PCR was optimized to run under the initial incubation conditions of 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 60°C for 1 min, for 45 cycles. The expression of the BDNF gene was normalized with the Glyceraldyehyde-3-phosphate Dehydrogenase (GAPDH) with ΔΔCT correlation.

Figure 2.
Figure 2.

Cell culture plate placed on SMS device.

Figure 3.
Figure 3.

Cell viability of SH-SY5Y cells exposed to different intensities of SMS. Cells were exposed to 0.1 T, 0.2 T, 0.3 T SMS for 60 min and analyzed using MTT. Results are presented in nm. Results are presented in nm. Data are expressed as medians (interquartile 25; interquartile 75) (Kruskal–Wallis, p>0.05).

Statistical analysis. The first analyses were normality and lognormality to choose between a parametric or non-parametric test. For parametric samples, data are presented as mean±SD and analyzed using Student's t-test. For non-parametric samples, data are presented as median (interquartile 25; interquartile 75) and analyzed using Kruskal–Wallis followed by Mann–Whitney test. Values of p<0.05 were considered significant. All analyses were performed using the statistical software GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

Results

Cell viability and cell death. MTT was performed to evaluate the SMS effects on the cell viability (Figure 3). SH-SY5Y cells were stimulated with SMS for 60 min in three different intensities (0.1 T, 0.2 T and 0.3 T). There was no significant difference between control cells and cells exposed to the three different SMS intensities (0.1 T, 0.2 T and 0.3 T) (Kruskal–Wallis, p>0.05). Based on these results, the highest intensity (0.3 T) and longer exposure time (24 h) were chosen for further analysis of cell viability (Figure 4). SH-SY5Y cells, evaluated immediately after 24 h of SMS exposure, presented a significant decrease in viability when compared to the control group (Student's t-test, p<0.05). Stimulated SH-SY5Y cells evaluated 24 h after SMS exposure did not present a significant difference in viability when compared to the control group (Kruskal–Wallis p>0.05). In differentiated SH-SY5Y, adipose-derived mesenchymal and HMVII cells, no difference in viability was found after SMS exposure for both evaluated periods (immediately and after 24 h of SMS exposure) (Student's t-test or Kruskal–Wallis, p>0.05).

Figure 4.
Figure 4.

Cell viability of SH-SY5Y, differentiated SH-SY5Y, HMV II and MSCh cells exposed to 0.3 T (305 mT) SMS. MTT analysis was done immediately after SMS exposure (24 h) and 24 h after SMS exposure (48 h). Results are presented as nm. In the MSCh, SH Dif and SH groups, data are expressed as mean±SD. In the HMV II group, data are expressed as medians (interquartile 25; interquartile 75). *Indicates significant difference when compared to the 24 h (-SMS) group (Student's t-test, p<0.05).

Figure 5.
Figure 5.

Cell death of SH-SY5Y, differentiated SH-SY5Y, HMVII and MSCh cells evaluated by PI/HO staining, immediately after SMS exposure (24 h) or 24 h after SMS exposure (48 h). Results are presented as percentages. In all groups, data are expressed as mean±SD. There was no difference between groups (Student's t-test, p>0.05).

In SH-SY5Y cells, PI/HO analysis of cell death (Figure 5) showed no significant difference after SMS exposure, suggesting there was no increase in cell death in these cells (Student's t-test or Kruskal–Wallis, p>0.05). Similarly, in differentiated SH-SY5Y no difference was found (Student's t-test or Kruskal–Wallis p>0.05). Annexin-V/PI evaluation (Figure 6) showed a decrease in apoptotic (Annex+/PI) (1.594% to 0.004%, Figure 6B) and double-positive (Annex+/PI+) cells (0.086% to 0.190%, Figure 6D), and an increase in necrotic (Annex-/PI+) (0.359% to 1.580%, Figure 6C) SH-SY5Y cells exposed to SMS for 24 h and analyzed 24 h after the stimulation (48 h) (Kruskal–Wallis, p<0.05, Figure 6). In groups evaluated immediately after 24 h of exposure to SMS (24 h), there was a decrease in double-positive (Annex+/PI+) cells (0.150% to 0.130%, Figure 6D).

Cell cycle (PI staining). Cell cycle analysis (Figure 7) of SH-SY5Y cells showed no difference in the percentage of cells in sub-G1, G1, S, G2 and >4N phases in both periods analyzed (Student's t-test, p>0.05), suggesting that exposure to SMS does not alter cell cycle distribution.

Figure 6.
Figure 6.

Cell death of SH-SY5Y cells evaluated by Annexin-V/PI staining, immediately after SMS exposure (24 h) or 24 h after SMS exposure (48 h). A) Live cells (Annex-/PI−). B) Apoptotic cells (Annex+/PI−). C) Necrotic cells (Annex-/PI+). D) Double-positive cells (Annex+/PI+). *Indicates significant difference when compared to the 24 h (-SMS) group (Kruskal–Wallis, p<0.05). #Indicates significant difference when compared to the 48 h (-SMS) group (Student's t-test, p<0.05). Data are expressed as mean±SD/Data as expressed as median (interquartile 25; interquartile 75).

Figure 7.
Figure 7.

Cell cycle analysis of SH-SY5Y cells evaluated by PI staining, immediately after SMS exposure (24 h) or 24 h after SMS exposure (48 h). Results are presented as percentages. Data are expressed as mean±SD. There was no difference between the different times (Student's t-test, p>0.05).

BDNF expression. In SH-SY5Y cells, even though detection of BDNF was successful (Figure 8), there was no difference in cells exposed to SMS for both periods when compared to control groups (Kruskal–Wallis, p>0.05). Although there was no difference in BDNF expression between the stimulated cells and the control, there was a difference between the stimulated groups, which is expected, due to analysis in different periods.

Figure 8.
Figure 8.

Gene expression analysis of BDNF gene in SH-SY5Y cells after SMS exposure (24 h) or 24 h after SMS exposure (48 h). Data are expressed as median (interquartile 25; interquartile 75). @Indicates significant difference between cells exposed to 24 h of SMS and cells exposed to 48 h of SMS (Kruskal–Wallis, p<0.05).

Discussion

Our results demonstrated that SH-SY5Y cells exposed to SMS for 24 h show a decrease in cell viability immediately after the exposure (24 h). A previous study using glioblastoma cells submitted to SMS for 24 h corroborates these findings regarding cell viability (15). This effect on cell viability, however, was not long-lasting, since 24 h after exposure treated groups were not different from the control group. SH-SY5Y cells did not show alterations in cell viability after exposure to magnetic stimulation, emphasizing that magnetic stimulation has cell type-dependent effects on cell viability.

PI/HO and Annexin-V/PI staining were performed in order to evaluate cell death. Evaluation of cell death 24 h after SMS exposure showed small differences compared to control cells (showing a decrease in apoptotic and double-positive cells, associated with an increase in necrotic cells). Alongside cell death, cell cycle profiling was performed, which indicated no changes in cell cycle distribution.

Our results indicated that SMS effects may also extend beyond the modulation of neuronal proliferation and plasticity. Neurotrophins, such as the brain-derived growth factor (BDNF), regulate the plasticity of the nervous system and are overexpressed in several types of cancer (27, 28). In fact, BDNF was initially characterized in oncogenic neuroblastoma, a type of cancer in nervous tissue (28). Even though the effects of SMS on nerve cells and brain tissues have been extensively described (23, 29-37), we found no difference in BDNF expression in SH-SY5Y cells exposed to SMS with the exception of an expected difference from 24 to 48 h.

When comparing our findings on undifferentiated and differentiated nerve cell lines only undifferentiated SH-SY5Y cells were influenced by SMS. Both SH-SY5Y cell subsets show differences ranging from polarization, number and length of the processes to proliferation (21, 38, 39), which may be distinctively affected by SMS. The difference in cell viability responses to SMS may not be due to the selectivity of action upon excitability (23) or other membrane channel-related effects (18, 40, 41), but may also influence other cellular processes. Indeed, SMS induces alterations in the viability of SH-SY5Y cells in response to cisplatin (24), having a modulatory effect on the cell's response to several pharmacological treatments, (42-47). Other effects of exposure to SMS have already been described in other cellular functions, such as ROS production (24), modulation of redox-related enzymes (25), pro- and anti-inflammatory cytokine release (24) and improvement in the killing function of NK cell (17). Given the diversity of the processes affected by SMS, changes in cell viability probably involve processes in addition to cell death, cell cycle distribution and neurotrophin production. Future studies, using higher intensities, as well as a longer exposure times, are necessary to evaluate if this technique induces or inhibits cell death.

Conclusion

The different effects exerted by exposure to SMS provide valuable information regarding the application potential of SMS. This study demonstrated that, considering the analyzed parameters, SMS is a potentially safe technique, at least in the utilized protocol (0.3 T SMS/24 h). The decrease in SH-SY5Y cell viability also shows potential for treatment of neuronal tumors with SMS. Also, our results showed that the effect of SMS is cell type specific. It is important to note that this is one of the first studies showing SMS as a potential tool in the treatment of neuronal tumors. Further investigations in this area are still necessary to better understand the effects of SMS exposure on cultured cells and in vivo models.

Acknowledgements

The Authors were supported by the Brazilian's agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (Dra ILS Torres; Dr. W. Caumo, Dr. P. R. S. Sanches), Federal University of Rio Grande do Sul and Brazilian Federal Agency for Support and Evaluation of Graduate Education – CAPES (Helouise Medeiros). The Authors also want to thank the Engineering Division from the HCPA for having developed the SMS stimulator; the Graduate Research Group of the Hospital de Clínicas de Porto Alegre – GPPG (I.L.S. Torres, grant number 15-0567); MCT/FINEP – COENG/2013. PRONEM FAPERGS. The Authors are also thankful for Research Incentive Fund received from FIPE/HCPA.

Footnotes

  • Authors' Contributions

    HRM, FSOO, EOCL, PRSS, FF, WC and ILST were responsible for the study concept and design. HRM, JA, MS, LN, NAACH and CUC contributed to the acquisition of the data. HRM, JA, CUC, FSOO, EOCL and ILST were responsible by data analysis. HRM, LFM, JA and ILST drafted the manuscript. All Authors revised and edited the manuscript and approved the final version.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare regarding this study.

  • Received June 30, 2020.
  • Revision received July 17, 2020.
  • Accepted July 20, 2020.

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Static Magnetic Stimulation Induces Cell-type Specific Alterations in the Viability of SH-SY5Y Neuroblastoma Cell Line
HELOUISE R. MEDEIROS, JOSÉ A. F. ASSUMPCAO, LICIANE F. MEDEIROS, MARTINA STAPENHORST, LARA NUNES, NICOLE A.C. HENCKES, CAROLINA URIBE CRUZ, FELIPE FREGNI, PAULO R.S. SANCHES, FERNANDA S.O. OLIVEIRA, WOLNEI CAUMO, ELIZABETH O. CIRNE-LIMA, IRACI L.S. TORRES
Anticancer Research Sep 2020, 40 (9) 5151-5158; DOI: 10.21873/anticanres.14518
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