Medical/biological study (experimental study)

ELF-MF attenuates quercetin-induced apoptosis in K562 cells through modulating the expression of Bcl-2 family proteins med./bio.

By: Brisdelli F, Bennato F, Bozzi A, Cinque B, Mancini F, Iorio R

Published in: Mol Cell Biochem 2014; 397 (1-2): 33-43

Aim of study (acc. to author)

To examine the effects of exposure to an extremely low frequency magnetic field on the apoptosis induced by four different compounds (vinblastine, etoposide, quercetin, resveratrol) in human K562 leukemia cells.

Background/further details

In the first experiment, the effects of 1 µM vinblastine, 1 µM etoposide (used as an anti-cancer drug) and 25 µM resveratrol (a stilbenoid, potential positive health effects are discussed) either alone or in combination with an extremely low frequency magnetic field on the growth and cell viability were examined.
In the second experiment, the effects of 25 µM quercetin (a flavonoid, has been suggested as an anti-cancer drug) either alone or in combination with an extremely low frequency magnetic field on the growth, cell viability, generation of reactive oxygen species, protein expression, enzyme activity of the caspase-3, apoptosis rate and cell cycle were examined.
In the third experiment, cells were exposed to different magnetic flux densities alone or in combination with quercetin and the number of apoptotic cells was determined.
Positive controls were performed.

Endpoint

cell viability/cell division/proliferation: apoptosis, cell viability, cell cycle distribution, generation of reactive oxygen species

Exposure

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: continuous for 24, 48 or 72 hours experiment 1 + 2	magnetic flux density: 1 mT
Exposure 2: 50 Hz Exposure duration: continuous for 24, 48 or 72 hours experiment 3	magnetic flux density: 0.01 mT magnetic flux density: 0.1 mT magnetic flux density: 2 mT

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 24, 48 or 72 hours
Additional info	experiment 1 + 2

Exposure setup

Exposure source	Helmholtz coils
Chamber	cells were exposed in petri dishes or multiwell plate
Setup	exposure system consisted of two identical apparatuses, each containing a waveform generator, a current amplifier, and a pair of Helmholtz coils; pair of Helmholtz coils had a mean radius of 13.0 ± 0.5 cm; in each coil, the number of turns was 800 with a 2 mm² wire giving a resulting resistance of 2.4 ohm and an inductance of 39 ± 1 mH; mean vertical distance between the coils was 13.5 ± 0.5 cm; current could either flow in the same direction (exposure) or in the opposite direction (sham system), where the magnetic flux density is "theoretically zero"; each coil was placed within two separate incubators (one for exposure, one for sham exposure) at a temperature of 37°C and an atmosphere of 95% air/5% CO₂and 100% relative humidity.
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	1 mT	-	measured	-	-

Additional parameter details

ambient DC field within the incubators was found to be 25.5 and 32.8 µT, respectively

Exposure 2

Main characteristics

Frequency	50 Hz
Type	magnetic field
Waveform	sinusoidal
Exposure duration	continuous for 24, 48 or 72 hours
Additional info	experiment 3

Exposure setup

Exposure source	same setup as exposure 1

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	0.01 mT	-	measured	-	-
magnetic flux density	0.1 mT	-	measured	-	-
magnetic flux density	2 mT	-	measured	-	-

Reference articles

Iorio R et al. (2013): ELF-MF transiently increases skeletal myoblast migration: possible role of calpain system
Kirschvink JL (1992): Uniform magnetic fields and double-wrapped coil systems: improved techniques for the design of bioelectromagnetic experiments

Exposed system:

intact cell/cell culture
K562 cells (human erythroleukemic cell line)

Methods Endpoint/measurement parameters/methodology

molecular biosynthesis: level of intracellular reactive oxygen species (oxidation reaction with dichlorofluorescein, spectrofluorometry); protein expression of Bcl-2, Bcl-xL, Mcl-1 and Hsp70 (Western blot)
cell viability/cell division/proliferation: cell proliferation (hemocytometer); cell viability (trypan blue exclusion assay); apoptosis (staining with acridine orange and ethidium bromide, fluorescence microscopy), apoptosis (Annexin V/FITC-staining, flow cytometry); cell cycle distribution (staining with propidium iodide, flow cytometry)
enzyme activity of caspase-3 (fluorescence staining, spectrofluorometry)

Investigated system:

isolated bio./chem. substance
intact cell/cell culture
cell extracts

Time of investigation:

after exposure

Main outcome of study (acc. to author)

In the first experiment, no significant differences were observed in the cell proliferation and cell viability between the exposed and the sham exposed cells. Vinblastine, etoposide, and resveratrol had an anti-proliferative effect on the cells. However, this effect was not influenced by the magnetic field exposure.
The second experiment showed that quercetin had an anti-proliferative effect, but this effect was attenuated by the magnetic field exposure: In cell cultures cultivated in the presence of quercetin and exposed for 48 and 72 hours, the cell viability was significantly increased and the number of apoptotic cells (also after 24 hours and as experiment 3 showed also at magnetic flux densities of 0.01 mT and 0.1 mT, but not at 2 mT) as well as the enzyme activity of the caspase-3 were significantly decreased compared to sham exposed cell cultures with quercetin. Additionally, in 48 hours-exposed cells with quercetin the numbers of cells in the G1 phase and the S phase were significantly increased and the number of cells in the G2 phase/mitosis significantly decreased compared to sham exposed cells with quercetin. The simultaneous treatment with magnetic field exposure for 24 hours and quercetin significantly increased the protein expression of Bcl-2 in comparison to the other groups and prevented the quercetin-induced significant down-regulation of Mcl-1 and Bcl-xL. After 48 hours of exposure, the protein expression of Hsp70 was significantly increased when compared to the control group, while after 72 hours it was significantly increased when simultaneously treated with exposure and quercetin. No significant changes occurred regarding the level of the reactive oxygen species.
The results indicate that extremely low frequency magnetic fields could be able to render K562 cells resistant to quercetin-induced apoptosis. Hence, these magnetic fields may have negative implications for anti-tumor therapies.

Study character:

medical/biological study
experimental study
full/main study
blind study

Study funded by

Ministero dell' Università e della Ricerca (MIUR (formerly MURST (Ministero dell' Università e della Ricerca Scientifica e Tecnologica); Ministry of University and Research), Italy

Ding Z et al. (2017): Vitamin C and Vitamin E Protected B95-8 and Balb/c-3T3 Cells from Apoptosis Induced by Intermittent 50Hz ELF-EMF Radiation
Feng B et al. (2016): Mitochondrial ROS Release and Subsequent Akt Activation Potentially Mediated the Anti-Apoptotic Effect of a 50-Hz Magnetic Field on FL Cells
Lee HC et al. (2015): Effect of extremely low frequency magnetic fields on cell proliferation and gene expression
Basile A et al. (2011): Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells
Garip AI et al. (2010): Effect of ELF-EMF on number of apoptotic cells; correlation with reactive oxygen species and HSP
Koh EK et al. (2008): A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species
Huang L et al. (2006): Effects of sinusoidal magnetic field observed on cell proliferation, ion concentration, and osmolarity in two human cancer cell lines
Palumbo R et al. (2006): Effects on apoptosis and reactive oxygen species formation by Jurkat cells exposed to 50 Hz electromagnetic fields
Santini MT et al. (2005): A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field
Ding GR et al. (2004): Extremely low frequency magnetic fields and the promotion of H2O2-induced cell death in HL-60 cells
Verheyen GR et al. (2003): Effect of coexposure to 50 Hz magnetic fields and an aneugen on human lymphocytes, determined by the cytokinesis block micronucleus assay
Pirozzoli MC et al. (2003): Effects of 50 Hz electromagnetic field exposure on apoptosis and differentiation in a neuroblastoma cell line
Yoshizawa H et al. (2002): No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines
Tofani S et al. (2001): Static and ELF magnetic fields induce tumor growth inhibition and apoptosis
Loberg LI et al. (2000): Cell viability and growth in a battery of human breast cancer cell lines exposed to 60 Hz magnetic fields

ELF-MF attenuates quercetin-induced apoptosis in K562 cells through modulating the expression of Bcl-2 family proteins med./bio.

Aim of study (acc. to author)

Background/further details

Endpoint

Exposure

Exposure 1

Main characteristics

Exposure setup

Parameters

Additional parameter details

Exposure 2

Main characteristics

Exposure setup

Parameters

Reference articles

Methods Endpoint/measurement parameters/methodology

Main outcome of study (acc. to author)

Study funded by

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