Alternative magnetic field exposure suppresses tumor growth via metabolic reprogramming

Abstract

Application of physical forces, ranging from ultrasound to electric fields, is recommended in various clinical practice guidelines, including those for treating cancers and bone fractures. However, the mechanistic details of such treatments are often inadequately understood, primarily due to the absence of comprehensive study models. In this study, we demonstrate that an alternating magnetic field (AMF) inherently possesses a direct anti-cancer effect by enhancing oxidative phosphorylation (OXPHOS) and thereby inducing metabolic reprogramming. We observed that the proliferation of human glioblastoma multiforme (GBM) cells (U87 and LN229) was inhibited upon exposure to AMF within a specific narrow frequency range, including around 227 kHz. In contrast, this exposure did not affect normal human astrocytes (NHA). Additionally, in mouse models implanted with human GBM cells in the brain, daily exposure to AMF for 30 min over 21 days significantly suppressed tumor growth and prolonged overall survival. This effect was associated with heightened reactive oxygen species (ROS) production and increased manganese superoxide dismutase (MnSOD) expression. The anti-cancer efficacy of AMF was diminished by either a mitochondrial complex IV inhibitor or a ROS scavenger. Along with these observations, there was a decrease in the extracellular acidification rate (ECAR) and an increase in the oxygen consumption rate (OCR). This suggests that AMF-induced metabolic reprogramming occurs in GBM cells but not in normal cells. Our results suggest that AMF exposure may offer a straightforward strategy to inhibit cancer cell growth by leveraging oxidative stress through metabolic reprogramming.

Keywords: alternating magnetic field (AMF); glioblastoma multiforme (GBM); metabolic reprogramming; mitochondria; oxidative phosphorylation (OXPHOS); reactive oxygen species (ROS).

FIGURE 1

Configuration and measured and simulated magnetic flux density B in the coil. (A) Photograph of a solenoid coil and the AMF generator (left). Photograph showing a solenoid coil wrapped in insulation tape (right). (B) Details of the shape and size of the solenoid coil. (C) Simulated magnetic flux density distribution in the coil cross‐section (AMF: 227 kHz, 250 Arms). The color scale represents magnetic flux density values, with red indicating high density and blue indicating low density. (D) Comparison of measured and simulated magnetic flux density B along the coil axis (cm). This schematic represents simulation values, with solid lines indicating the simulated data. The red dots denote actual measured values. (E) Simulated magnetic flux density B in the radial direction (cm) of the coil. This schematic represents simulation values, with solid lines indicating the simulated data.

FIGURE 2

Suppression of cancer cell proliferation by AMF at 227 kHz for more than 30 min. (A) The effect of different frequencies (kHz) of AMF (250 Amrs) on the proliferation of GB cell lines (U87 and LN229). XTT cell proliferation assays were conducted at various AMF frequencies (kHz) for 30 min, with evaluation occurring 24 h post‐AMF exposure (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001 vs. 0 kHz). (B) The impact of varying electric current intensities (Arms) in AMF (227 kHz) on the proliferation of GBM cell lines (U87 and LN229) (n = 4, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. 0 Arms). (C) The effect of different exposure durations (min) to AMF (227 kHz, 250 Amrs) on the proliferation of GBM cell lines (LN229, U251) (n = 4, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. 0 min). (D) The influence of AMF (227 kHz, 250 Amrs) on other GB cell lines (U251, T98, and A172), a pancreatic cell line (PANC1), human breast cancer cell lines (MCF7, MDA‐MB‐231, MDA‐MB‐453), normal human astrocyte (NHA), human cardiac fibroblast (HCF), and human umbilical vein endothelial cells (HUVEC) (n = 4, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL). (E, F) Continuous monitoring of cell growth with and without a 30‐min AMF exposure (227 kHz, 250 Arms) in U251 and LN229 cell lines. In vitro cell proliferation was measured using the xCELLigence Real‐Time Cellular Analysis system. (G) Cell cycle analysis 3 and 24 h post‐AMF exposure (227 kHz, 250 Arms, 30 min), revealing the inhibitory effect of AMF, notably the induction of S and G2 phase arrest (n = 4, ns, not significant, **p < 0.01, ***p < 0.001 vs. CTRL). (H) Immunoblot analysis of phosphorylated and unphosphorylated forms of p53, p21, CDK2, Cyclin A, Cyclin B1, Cyclin D1, Cyclin E, and GAPDH 24 h after a 30‐min AMF exposure (227 kHz, 250 Arms) (n = 4, ns, not significant, **p < 0.01, ***p < 0.001 vs. CTRL).

FIGURE 3

Anti‐cancer effects of AMF in mice subcutaneous and brain GBM models. (A) Changes in the volume (mm³) of subcutaneous tumors (U87 and LN229 cells) over 14 days in the control group versus the AMF treatment (227 KHz, 250 Amrs, 30 min per session) group. (B) Photographs of tumors from subcutaneous implantation of U87 cells (control and AMF treatment groups). (C) Representative images of brain tumor sections following H&E staining and Ki‐67 staining. The left images are from the control group, and the right images are from the AMF treatment group (n = 4). The graph shows the ratio of Ki‐67 positive cells in both the control and AMF treatment groups. White arrows indicate positive area. Calibration bar: 500 μm. (D) Schedule of AMF treatment for the mouse brain GBM model: 227 kHz, 250 Arms, 30 min per session, 5 times per week for a duration of 2 weeks. (E) Representative images from the in vivo imaging system (IVIS) images of mouse brains at 6, 11, 16, and 21 days post‐implantation of U87 cells. (F) Luminescent intensity comparison between the control and AMF treatment groups. The graph depicts the time course of tumor volume changes (n = 6). (G) Overall survival curve of mice in the study. The blue line represents the survival percentage (%) in the AMF‐treated group, while the black line represents survival in the control group (without AMF treatment).

FIGURE 4

Comprehensive analysis of protein expression and phosphorylation induced by AMF in GBM cells. (A) Timeline for conducting the microarray analysis. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis illustrating pathways that are enriched with differentially expressed genes. Comparison made between proteins collected from U87 cells exposed to 30 min of AMF (250 kHz, 250 Arms) and those from U87 control cells (without AMF). (C) Timeline for the iTRAQ phosphorylated protein analysis. (D) Changes in protein expression induced by AMF (250 kHz, 250 Arms) in U87 cells. Histograms display the count of proteins whose phosphorylation levels were either upregulated (red) (>1.5‐fold) or downregulated (blue) (<1.5‐fold) following AMF exposure at 0.5, 1, and 2 h. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing pathways enriched with differentially expressed genes. Comparison made between proteins collected from U87 cells 0.5 h post‐AMF exposure and proteins from GBM cells not exposed to AMF.

FIGURE 5

AMF promotes ROS production and increases MnSOD. (A) Increase in mitochondrial membrane potential in U87 cells following 30 min of AMF exposure (n = 4, ns, not significant, *p < 0.05, ***p < 0.001 vs. CTRL). (B) Measurement of ROS production in U87 cells at 0, 24, and 48 h post 30‐min AMF exposure (n = 4, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL). (C) ROS production in U87 cells treated with 10 μM potassium cyanide (KCN) following 30 min of AMF exposure (n = 4). (D) ROS production in U87 cells treated with 5 mM NAC following 30 min of AMF exposure (n = 4). (E) Immunoblot analysis of MnSOD, Cu/ZnSOD, and cytochrome c protein expression in GBM cells (LN229) exposed to AMF for 30 min at various time points (0, 5, 15, 30 min, 1, 6, 12, 24, 48 h) (n = 4). (F) Immunoblot analysis of MnSOD phosphorylation and protein expression 6 h post‐AMF exposure for 30 min, with or without 10 μM KCN in LN229 cells (n = 4). (G) Immunoblot analysis of MnSOD phosphorylation and protein expression 6 h post‐AMF exposure for 30 min, with or without 5 mM NAC in LN229 cells (n = 4). In all these experiments, the AMF was conducted under conditions of 227 kHz and 250 Arms.

FIGURE 6

AMF promotes mitochondrial biogenesis. (A) Real‐time PCR analysis of mitochondrial DNA (mtDNA) at 6, 24, and 48 h post 30‐min AMF exposure in LN229 (n = 4, ns, not significant, *p < 0.05 vs. CTRL). (B) Immunoblot analysis of PGC1α protein levels at 15 min, 3, 6, 12, and 24 h following 30 min of AMF exposure in LN229 (n = 4, *p < 0.05, ***p < 0.001 vs. CTRL). (C) Immunoblot analysis of PGC1α protein levels in the presence of 10 μM potassium cyanide (KCN) 6 h post 30‐min AMF exposurein LN229 (n = 4). (D) Immunoblot analysis of PGC1α protein levels in the presence of 5 mM NAC 6 h after 30 min of AMF exposure in LN229 (n = 4). In all these experiments, the AMF was conducted under conditions of 227 kHz and 250 Arms.

FIGURE 7

Metabolic reprogramming in GBM cells induced by AMF. (A) Extracellular acidification rate (ECAR) levels of GBM cells (LN229) measured after 30 min of AMF exposure. (B) Oxygen consumption rate (OCR) levels of GBM cells following 30 min of AMF exposure (C) ECAR levels of GBM cells under different kHz conditions immediately after 30 min of AMF exposure. (D) OCR levels of GBM cells at various kHz conditions immediately following 30 min of AMF exposure . (E) ECAR levels of GBM cells at different intensities (Arms) immediately after 30 min of AMF exposure. (F) OCR levels of GBM cells at various intensities (Arms) immediately following 30 min of AMF exposure. (G) ECAR levels of GBM cells treated with 10 μM KCN after 30 min of AMF exposure . (H) OCR levels of GBM cells in the presence of 10 μM KCN following 30 min of AMF exposure. (I) ECAR levels of GBM cells treated with 5 mM NAC after 30 min of AMF exposure. (J) OCR levels of GBM cells in the presence of 5 mM NAC following 30 min of AMF exposure (n = 4, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL). (K) Proposed mechanism of AMF‐mediated cell cycle alteration in GBM cells. In all these experiments, the AMF was conducted under conditions of 227 kHz and 250 Amrs,