Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms

Abstract

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H₂O₂ and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

Figure 1

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the major ROS-targeting antioxidant enzymatic defence systems in SH-SY5Y human neuroblastoma cells. Glutathione peroxidase (tGPX)/total superoxide dismutase (tSOD) (panel a) and catalase (CAT)/total superoxide dismutase (tSOD) (panel b) ratios were reported. Empty histograms represent unexposed cells (sham), whereas gray histograms represent ELF-MF-exposed cells. Values were expressed as means ± s.d. ***P < 0.001 (2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison).

Figure 2

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the levels of oxidatively modified proteins (panel a) and DNA (panel b) in SH-SY5Y human neuroblastoma cells. Empty histograms represent unexposed cells (sham), whereas gray histograms represent ELF-MF-exposed cells. Representative photographs of alkaline comets observed through fluorescence microscopy were reported in panel b (bar = 30 µm). Protein carbonyls were expressed as means ± s.d. Percentages of DNA in the comet tails were expressed as medians with interquartile ranges. *P < 0.05, **P < 0.01 (2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison). ***P < 0.001 (Mann-Whitney rank sum tests).

Figure 3

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the protein levels of SIRT1 (panel a) and SIRT3 (panel b) in SH-SY5Y human neuroblastoma cells. Empty histograms represent unexposed cells (sham), whereas gray histograms represent ELF-MF-exposed cells. Representative grouping of Western blots were reported in both panels, with clear white space delineation between the proteins of interest (i.e., SIRT1 and 3) and the loading control (i.e., β-actin). T5 and T10, exposed cells for 5 and 10 days, respectively (C5 and C10, time-matched unexposed controls). Values were expressed as means ± s.d. *P < 0.05, ***P < 0.001 (2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison).

Figure 4

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the protein levels of NRF2-phosphoS40 in SH-SY5Y human neuroblastoma cells. Empty histograms represent unexposed cells (sham, CTR), whereas gray histograms represent ELF-MF-exposed (TRT) cells. Representative grouping of Western blots were reported in panel a, with clear white space delineation between the protein of interest (i.e., NRF2-phosphoS40) and the loading control (i.e., β-actin). T5 and T10, exposed cells for 5 and 10 days, respectively (C5 and C10, time-matched unexposed controls). Representative photographs of immunofluorescence microscopy were reported in panel b (bar = 40 µm). Western blotting-related results were expressed as means ± s.d. Immunofluorescence-related results were expressed as medians with interquartile ranges. *P < 0.05, **P < 0.001 (2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison). ***P < 0.001 (Mann-Whitney rank sum tests).

Figure 5

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the survival of SH-SY5Y neuroblastoma cells after a 2-h incubation with 35 μM H₂O₂. Values were expressed as means ± s.d. A 20% decrease of alive cells (IC₂₀) was calculated through a 4P-logistic regression derived from a dose-response curve obtained by incubating unexposed cells with H₂O₂ concentrations ranging from 0 to 200 µM (inset diagram). ***P < 0.001 (2 × 2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison).

Figure 6

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on the survival of SH-SY5Y neuroblastoma cells after a 48-h incubation with 90 nM doxorubicin (DOXO). The concentration of doxorubicin was chosen on the basis of the reported continuous steady-state concentration achieved in blood during clinical treatments of children cancers^, . Values were expressed as means ± s.d. ***P < 0.001 (2 × 2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison). For data derived from the 100 µT ELF-MF exposure, please refer to the 3.6 Results section.

Figure 7

Effects of 5 or 10 days of ELF-MF exposure (1 mT, 50 Hz) on one of the major detoxification enzymatic defence systems in SH-SY5Y human neuroblastoma cells. The specific activity of glutathione S-transferase (GST) was reported. Empty histograms represent unexposed cells (sham), whereas gray histograms represent ELF-MF-exposed cells. Values were expressed as means ± s.d. *P < 0.05, ***P < 0.001 (2 × 2 factorial ANOVA followed by post-hoc Newman–Keuls/Tukey tests for multiple comparison).