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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Cancer. 2014 Jul 1;120(21):3418–3425. doi: 10.1002/cncr.28895

Non-invasive radiofrequency treatment effect on mitochondria in pancreatic cancer cells

Steven A Curley a,b, Flavio Palalon a, Xiaolin Lu a, Nadezhda V Koshkina a,*
PMCID: PMC4365904  NIHMSID: NIHMS669657  PMID: 24986120

Abstract

Background

Development of novel therapeutic approaches for cancer therapy is important, especially for tumors that have poor response or develop resistance to standard chemotherapy and radiation. We discovered that non-invasive radiofrequency (RF) fields can affect cancer, but not normal cells, inhibit progression of tumors in mice, and enhance anticancer effect of chemotherapy. However, it remains unclear what physiological and molecular mechanisms this treatment induces inside cells. Here, we studied the effect of RF treatment on mitochondria in human pancreatic cancer cells.

Methods

Morphology of mitochondria in cells was studied by electron microscopy. Alteration of mitochondrial membrane potential (Δψ) was accessed with Mitotracker probe. Respiratory activity of mitochondria was evaluated by changes in oxygen consumption rates (OCR) determined with MitoStress kit. Production of intracellular reactive oxygen species (ROS) was performed using flow cytometry. Colocalization of mitochondria and autophagosome markers in cells was done by fluorescence immunostaining and confocal microscopy analysis.

Results

RF changed morphology of mitochondria in cancer cells, altered polarization of the mitochondrial membrane, substantially impaired mitochondrial respiration, and increased ROS production, which indicate on the RF-induced stress on mitochondria. We also observed frequent colocalization of the autophagosome marker LC3B with the mitochondrial marker Tom20 inside cancer cells after RF exposure indicating on the presence of mitochondria in the autophagosomes. This suggests that RF-induced stress can damage mitochondria and induce elimination of damaged organelle via autophagy.

Conclusion

RF treatment impaired the function of mitochondria in cancer cells. Therefore, mitochondria can represent one of the targets of the RF treatment.

INTRODUCTION

The effects of electromagnetic fields on cancer cells lead to discovery of major diagnostic tools such as X-ray imaging, computed tomography, and magnetic resonance imaging due to their ability to penetrate through the human body. However, the energy produced by these ionizing radiation devices is generated by short electromagnetic waves (micron to nanometer length) with very high frequency at 1015–1024 Hz (this range covers ultraviolet and gamma-rays) and is destructive not only for cancer, but for normal cells as well. Therefore, application of these electromagnetic fields is limited and can cause long-lasting toxic effects on vital organs, as happens in patients undergoing ionizing radiation treatment. Long electromagnetic waves within the range of meter, known as radiowaves, have low frequency (1010–1.0 Hz) and are produced by common home electronic devices such as radios and televisions. Electromagnetic fields produced by radiowaves are recognized to be safe for humans because they have low absorbance rates by human tissues and cells, when compared with those produced by short electromagnetic waves of high frequency1.

The ability of low energy electromagnetic waves to affect cancer cells was demonstrated in several in vitro studies2–7. Some studies verified their anticancer effect in vivo2,3,8–10 or were studied in patients2,11,12. Molecular changes that can be induced in cancer cells after exposure to the RF fields remain poorly understood. Most reports suggest that RF fields cause changes in the function of tubulin, the protein that plays an essential role in microtubule formation during cell division in cancer cells4–7. Several studies of the biological effects of electromagnetic fields with low-frequency on cells indicate other changes, such as altering the function of ion channels on cell plasma membranes13,14. In our recent studies, we reported that non-invasive RF treatment at 13.56 MHz with the non-invasive field ranging from 1 KeV to 20 KeV/m2 inhibits the growth of orthotopic hepatocellular carcinoma in mice15, and enhances the anticancer effect of gemcitabine chemotherapy in a murine model of orthotopic pancreatic cancer16. Investigation of RF-induced cell death mechanism in cells indicated on its ability to affect cancer cells via autophagy16. Importantly, this effect was not observed in normal cells. Autophagy is one of the mechanisms utilized by cells for processing damaged organelles. Mitochondria is one the most sensitive cellular organelle to external and internal stresses, which perhaps, explains the short life time and rapid turnover of this organelle. Even in non-proliferating tissues it constantly turns over with a half-life of approximately 10–20 days17. Autophagy of outworn or damaged mitochondria is known as mitophagy. Therefore, in this report we focused on studying the RF field effect on mitochondria and its function in cancer cells. Because we previously obtained positive results of this novel non-invasive method for the treatment of pancreatic cancer, one of the deadliest types of cancer with very limited therapeutic options, we used human pancreatic cancer cells in the current study. We discovered that our RF treatment caused a substantial change in morphology and function of mitochondria in cancer cells.

MATERIALS AND METHODS

Cell Culture

Human pancreatic cancer cells, AsPC-1 and Panc-1 were acquired from the American Type Culture Collection (Manassas, VA). Cells were maintained in standard growth conditions (37°C, 5% CO2), Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco,Grand Island, NY), and authenticated by the Characterized CellLine Core (STR DNA fingerprinting, M.D. Anderson Cancer Center).

RF Treatment

For in vitro studies cells were seeded at 0.1×106 cells/well in 2 ml of media into 12-well plates and after overnight incubation were exposed for 5 minutes to the RF field at 900W at a frequency of 13.56 MHz (Therm Med LLC, Erie, PA), as described elsewhere18. Before each experiment, the device was calibrated for heating profile using gold nanoparticles. After RF treatment, cells were placed back into the tissue culture incubator until the planned time for analysis. Cells that remained unexposed to the RF field were used as control.

High-resolution transmission electron microscopy imaging of autophagy in vitro

Cells were exposed to the RF field for 5 min and fixed on the following day with 1M cacodylate buffer (pH 7.4) containing 3% glutaraldehyde and 2% paraformaldehyde for 24 h. Samples were then washed with 0.1% cacodylate buffered tannic acid and treated with 1% osmium tetroxide. Finally, samples were stained with 1% uranyl acetate, dehydrated with ethanol, and embedded in LX-112 medium. After polymerization, samples were cut on a microtome and double stained with uranyl acetate/lead citrate. Imaging was done using a JEM1010 transmission electron microscope (Jeol USA, Inc. Boston, MA) equipped with the AMT Imaging System (Advanced Microscopy Techniques, Corp. Danvers, MA).

MitoTracker Staining

Prewarmed at 37°C, MitoTracker Red FM reagent (Molecular Probes, Inc., Eugene, OR) was added to cancer cells immediately after RF exposure at final concentration of 0.5 mM. Untreated cells were used as control. All cells were placed into the tissue culture hood for 30-min incubation. After that the cells were washed with warm PBS and fixed with 2% formaldehyde. Fixed cells were analyzed under an Olympus IX81 fluorescent microscope (Center Valley, PA). Quantitative analysis of fluorescence intensity was performed on three randomly selected microscopic fields with the use of Image-Pro software program (Media Cybernetics, Rockville, MA).

OCR Measurement

Seahorse XF96 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) provides real-time measurements of oxygen consumption rate (OCR) in cells. Mitostress kit that is used to measure basal respiration, ATP turnover, proton leak, and spare respiratory capacity was purchased from Seahorse Biosciences (Billerica, MA). Preparation of cells and analysis for this assay was performed according to the vendor instructions. Briefly, RF-treated cancer cells and untreated cells that were used as control were harvested, reconstituted in the equilibrated Assay Medium to a final concentration 0.1×106 cells/ml and seeded in quintuplets into 96-well cartridges. The cartridge with cells was placed for 30 min into the Seahorse XF96 Analyzer incubator unit before running a program for equilibration. Basal rates of oxygen consumption were measured four times during the first 35 min. If fluctuations in OCR levels during this time did not exceed 10%, the assay continued and other chemicals (Oligomycin, FCCP, and a mixture of Antimycin A with Rotenone) were subsequently added to cells. After two minutes of mixing, post-exposure OCR measurements were made 4 times.

ROS production measurement

Triplicates of cells (1× 106 cells/well seeded in 12-well-plate) were exposed to the RF field for 5 min or remained untreated and were trypsinized immediately after the end of treatment. To inhibit cytotoxic effect of trypsin cells were first resuspended in 1 ml of serum-containing culture media. After centrifugation media over the cells was substituted with 1 ml PBS containing 1 µg/ml of hydroethidine and 6 µg/ml of 2',7'-dichlorodihydrofluorescein diacetate were added to Panc-1 and AsPC-1 cells, respectively. Untreated unstained cells were used as a negative control. Cells were incubated for 30 min at 37°C. After washing with PBS cells were analysed on LSRII flow cytometer (BD Biosciences, San Jose, CA).

Fluorescent Immunocytochemistry Staining for Mitochondria and Autophagy Markers

Cells were fixed 5 minutes after RF exposure with 2% paraformaldehyde overnight and then stained with Tom20 (F10) antibody (Santa Cruiz Biotechnology, Inc., Dallas, TX) as a marker for mitochondria or anti-LC3B antibody (Cell Signaling Technology, Danvers, MA) as a marker of autophagosomes. Fluorescently labeled secondary antibodies were added to track the presence of the primary antibodies bound with the targets – FITC-labeled secondary antibody was used for binding with LC3B, Texas Red secondary antibody was used for binding with Tom20. Images of cells were taken using Olympus IX81 inverted fluorescent microscope (Olympus Inc., Center Valley, PA).

Statistics

Results from experiments are presented as means with standard deviations. GraphPad Instat 3 software (GraphPad Software Inc., La Jolla, CA) was used for evaluation of distribution assumption of analysis and validation of the test type for statistical analysis. All results showed normal pattern of values distribution and were analyzed by two-sided Student’s t-test. P<0.05 was considered statistically significant. Flow cytometry analysis involved data from at least 10 000 events that was repeated thrice and representative histograms were selected for publication.

RESULTS

Exposure of cancer cells to the RF field induced morphological changes of mitochondria

AsPC-1 and Panc-1 human pancreatic cancer cells were exposed to the RF field for 5 min and fixed 24 h after treatment as shown in Fig. 1. In our previous studies, we already observed the ability of RF treatment to change cellular morphology by causing shrinking and detachment of cancer cells, which was followed by significant reduction of their viability16. Here examination of cellular organelles under electron microscopy revealed the most significant morphological changes in mitochondria (Fig. 2). Many cells had enlarged bloated mitochondria with altered shape of cristae.

Figure 1. Cells treatment in the RF field.

Figure 1

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Cells were seeded in culture dishes and placed between the transmission top head and reciprocal bottom platform of the RF unit. Treatment was performed during 5 min at 13.56 MHz and at a power of 900W.

Figure 2. RF treatment altered the morphology of mitochondria in pancreatic cancer cells.

Figure 2

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Cells were fixed for the TEM imaging 24 h after RF treatment. Untreated cells were used as control. Mitochondria are indicated by black arrows.

RF treatment altered mitochondrial membrane potential (Δψ) in pancreatic cancer cells

To further validate the effect of RF field on mitochondria in cancer cells, we investigated whether it altered membrane polarization of mitochondria. For that we stained cells before and after RF exposure with Mitotracker Deep Red FM probe, which is a red-fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon membrane potential. Treatment of AsPC-1 cancer cells in the RF field for 5 min caused 6-fold elevation of red fluorescent signal from 6.2±3.2 to 48.2±12.3 (p=0.007), when compared with untreated cells (Fig. 3). In Panc-1 cells RF treatment was followed by 3-fold increase in fluorescence signal, from 7.7±0.7 to 23.6±10.8 (p=0.04), indicating an RF-induced alteration of Δψ.

Figure 3. MitoTracker Red fluorescent staining was elevated in pancreatic cancer cells after RF exposure.

Figure 3

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Cells were exposed to the RF field for 5 min. MitoTracker reagent was added immediately after the end of treatment and cells were incubated for 30 min. After washing, cells were fixed and imaged for fluorescence intensity in red spectrum.

RF treatment decreased oxygen consumption rates (OCR) in pancreatic cancer cells

We also analyzed the pattern of OCR in cancer cells in response to the RF-induced stress by MitroStress Kit which directly measures of the activity of electron transport chains in mitochondria. As seen in Fig. 4, RF treatment caused significant decline in the OCR levels from 103±12 pmoles/min in untreated AsPC-1 cells to 43±22 after treatment (p=0.0001). Similarly, in Panc-1 cells average OCR levels in untreated cells remained at 203+15 pmoles/min and reduced to 143+35 pmoles/min (P=0.0018) after RF exposure.

Figure 4. RF treatment decreased oxygen consumption rates (OCR) in pancreatic cancer cells.

Figure 4

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AsPC-1 cells (top panel) or Panc-1 cells (bottom panel) were harvested immediately at the end of 5-min RF field exposure and left for equilibration in the Seahorse ×96 incubator. OCR measurements were performed with the use of Mitostress kit and analyzed by the Seahorse Instrument Software as described in Materials and Methods.

The ATP coupler oligomycin is used to prevent phosphorylation respiration in cells. Treatment of cells with oligomycin permits evaluation of oxygen consumption levels devoted for ATP synthesis in cells. As expected, levels of OCR in cells that were not exposed to the RF treatment declined after exposure to oligomycin as shown on Fig. 4. RF treatment had some additive effect on oligomycin-induced decline of OCR in AsPC-1 cells and insignificant effect in Panc-1 cells, though in both cases exposure of cells to the RF caused reduction of ATP production.

The recovery of mitochondrial function after Oligomycin treatment can be achieved by addition of the uncoupling FCCP reagent. This agent is used to determine maximum respiration in cells. OCR levels in untreated and RF-treated cancer increased. However, addition of FCCP to cells that were exposed to the RF field did not allow achiev maximal respiration levels which were recorded for those cells that were not exposed to the RF. The average maximal OCR values in RF-treated cells after addition of FCCP reagent were 1.5-2-fold lower when compared with those in RF-untreated cells.

Finally, treatment of cells with Rotenone and Antimycin A causing functional arrest in mitochondrial complexes I and II, shut down mitochondrial respiration completely. Results on Fig. 4 demonstrate that RF treatment did not alter cells response to these reagents.

RF treatment stimulates ROS production in pancreatic cancer cells

Frequent response of mitochondria to stress is revealed by elevation of ROS production. Therefore, we used oxidant-sensing fluorescent probes to determine the levels of ROS production in pancreatic cancer cells after RF-induced stress. We observed the shift of mean fluorescence intensity from 1.2×103 in untreated AsPC-1 cells to 1.6×103 immediately after 5-min exposure to the RF (Fig.5). The same pattern was noticed in Panc-1 cells that showed fluorescence elevation from 1.3×103 in untreated state to 1.0×104 after RF treatment. Elevation of fluorescence intensity in both cases indicated on elevation of ROS production in cancer cells after RF exposure.

Figure 5. RF treatment enhanced ROS production in pancreatic cancer cells.

Figure 5

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AsPC-1 cells (top panel) or Panc-1 cells (bottom panel) were harvested immediately at the end of 5-min RF field exposure and treated with HE or DCFH-DA, respectively, as described in Materials and Methods. Stained cells were analyzed by flow cytometry.

RF treatment increased colocalization of mitochondria with autophagosomes

Previously we were able to show that RF treatment can induce autophagy in pancreatic cancer cells16. Damaged organelles, including mitochondria can be processed in cells via autophagy19. Data from our experiments indicate that RF treatment induced substantial stress on mitochondria, which may be damaging. The first step of processing damaged mitochondria is uptake inside autophagosome vesicles. This event in cells can be verified by fluorescent colocalization of LC3B as a marker for autophagosomes membrane with the mitochondria marker Tom20, the protein expressed on the outer membrane of mitochondria. Increased levels of LC3B green fluorescent puncta were noticed only in cells exposed to the RF field and indicate on increased presence of autophagosomes in cells after RF exposure (Fig. 6) and correlate with our previous studies where we performed a thorough investigation of RF-induced autophagy16. Red fluorescence staining with Tom20 antibody designating mitochondria was similar in both treated and untreated cells. Co-localization of the LC3B (red) and TOM20 (green) signals revealed negligible co-localization of Tom20 with LC3B in AsPC-1 and Panc-1 cells but large areas of co-localization (orange/yellow color) in these cells were observed after RF exposure, suggesting that autophagy-mediated mitochondrial degradation occurs in pancreatic cancer cells upon RF treatment.

Figure 6. RF treatment enhanced colocalization of damaged mitochondria with autophagosomes.

Figure 6

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Cells were treated in the RF field for 5 min and fixed after 48 h. Fluorescent immunocytochemical staining of cells was done with Tom20 antibody (red) as mitochondria marker and with LC3B antibody (green) as autophagosome marker. DAPI was used for nuclei staining (blue). Images were obtained using fluorescent confocal microscopy.

DISCUSSION

Mitochondria play an important role in different physiological and pathological processes in eukaryotic cells. Studies unraveling the detailed mechanisms of their function in cancer cells provided the unique targets for cancer cell suicide and lead to the development of the innovative class of anticancer drugs, the so called mitochondrion-targeted agents20. Multiple chemotherapeutic and non-chemotherapeutic modalities that cause cancer cell death were shown to disrupt the function of mitochondria by affecting their phosphorylating respiration as an initial step in the subsequent cascade of apoptosis-inducing events. In the current study we were able to demonstrate the ability of a novel non-invasive method of cancer treatment based on the use on RF fields to alter the function of mitochondria. Along with morphological changes that were observed in mitochondria of pancreatic cancer cells in response to RF treatment, we observed alteration of mitochondrial Δψ and a significant 1.5-2-fold decline of the oxygen consumption and ATP production. Finally, mitochondria is the major source of ROS production during cellular stress. During normal metabolism, ROS are produced in small amounts as byproducts and are quickly deactivated or reduced by special intracellular enzymes and small antioxidant molecules, thus avoiding harm to the cell. In stressful environments, synthesis of ROS can be increased dramatically and cause mitochondria damage followed by induction of programmed cell death. We demonstrated the induction of ROS production in pancreatic cancer cells in response to RF-induced stress. In our previous studies we observed that similar or lower doses of RF treatment were sufficient to induce cell death or inhibit proliferation in different types of malignant cells, including pancreatic and liver cancer cells in vitro and in vivo15,16. Results obtained in the current study suggest that cytotoxic effect of RF treatment may be, at least in part, mediated by affecting mitochondria function.

Recent studies by other investigators demonstrated that deprivation of ATP production can stimulate autophagy mechanism in cells for alternative energy sources21,22. In our previous studies we were able to show that RF treatment of cancer cells was followed by induction of autophagy16. Data obtained in the current study indicate that substantial reduction of oxygen consumption in RF-treated cancer cells could be one of the initiating signals for autophagy activation as seen by elevation of green LC3B puncta signal in Fig. 6. Moreover, exposure of mitochondria to the RF field could be devastating and lead to mitochondria damage followed by mitophagy in cancer cells. Increased colocalization of the mitochondria marker Tom20 with the autophagosome marker LC3B that we observed in cancer cells after RF treatment indicate the ability of RF treatment to cause mitochondria damage leading to elimination of these damaged structures by autophagosomes. We are now identifying mitophagy markers that elucidate this mechanism and will allow detailed understanding of the processing of mitochondria-containing autophagosomes in cancer cells after RF exposure.

In summary, results obtained in the current study demonstrate that RF treatment can affect mitochondria in cancer cells by weakening their oxygen consumption and ATP production and causing mitochondrial damage followed by induction of substantial co-localization of mitochondria in autophagic vesicles. This reveals mitochondria as a potential target in cancer cells for RF treatment and warrants further investigation.

ACKNOWLEDGEMENTS

We thank Rosalind Ramos for assistance in preparation of this manuscript for publication. We acknowledge Dr. Jared K. Burks and Kenneth Dunner, Jr. for assistance with fluorescent and electron microscopy. We also thank Dr. Elsa R. Flores and Dr. Xiaohua Su for training and sharing Seahorse equipment with us.

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