CC BY
4DOI 10.24412/cl-37136-2023-1-63-68
MODULATION OF CELLULAR RESPONSES TO IONISING RADIATION BY RADIOFREQUENCY FIELDS: POTENTIAL MECHANISMS
ANGELA CHINHENGO AND JOHN AKUDUGU
Division of Radiobiology, Department of Medical Imaging and Clinical Oncology, Stellenbosch
University, South Africa
jakudugu@sun.ac.za
ABSTRACT
Radiotherapy is one of the most used treatment options for different kinds of cancer and its main goal is to kill tumour cells while minimising toxicity normal tissue. A sure means of killing cancers is to increase the dose radiation that is administered to patients, but dose escalation may lead to adverse normal tissue toxicity. Hence, the need to develop noninvasive ways to sensitise tumour cells to low doses of radiation. Pharmaceuticals have been used to partly address this need, but systemic side effects pose another challenge. Electromagnetic fields (EMF) which largely thought to be nonhazardous have long been shown to have wide ranging benefits in the treatment of disorders as diverse as fractures, wounds, depression, and cancer [1- 3]. However, EMF have equally exhibited both desirable and undesirable effects and their anticancer and cancer-promoting properties have been demonstrated [4]. These effects have been observed for EMF frequencies ranging from fractions of a hertz (Hz) to several petahertz (PHz).
Specifically, radiofrequency fields (RF) have received significant attention in the clinic. For instance, they have been extensively used in radiofrequency ablation of hepatocellular carcinoma [5-7], early-stage renal cell carcinoma [8], non-small cell lung cancer [9], and melanoma [10-12]. Radiofrequency ablation typically employs a frequency of ~500 MHz which falls within the medium radio wave range, and the desired biological effect of this procedure is mediated by induction of molecular frictional heating in the target tissue by the radio wave [13]. The technique is minimally invasive (electrodes and probes inserted into target tissue) and is thought to be more superior than surgery. An informed combination of radiofrequency fields with radiotherapy could prove beneficial in cancer management. In a series of studies, we sought to evaluate the effect of low frequency radio waves on the sensitivity in vitro cell cultures to X-ray irradiation. The main objectives of these investigations were to confirm the capacity of RF to modulate cellular radiosensitivity and identify potential underlying mechanisms for such modulation. For this, Chinese hamster lung fibroblasts (V79) and human melanoma cells (MeWo) were remotely exposed to radiofrequency fields prior to or post irradiation with X-rays and processed for colony forming cell survival [14,15]. The radiofrequencies were generated from an EMEM oscillator amplifier (EMEM Devices Rife Machine, Model No. 1-2012B, Boulder, CO, USA; Fig. 1) with a carrier frequency of 27.125 MHz, square amplitude modulated at 100 and 1000 Hz. The modulating frequencies were injected from a GME frequency generator (GME Technology, Model No. SG-10, Panoma, CA, USA). In the cell cultures, the RF-induced magnetic flux densities were estimated to range between 0.05 and 0.25 ^T. The X-ray irradiation was performed using a Faxitron MultiRad 160 X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA). The irradiation was performed at a dose rate of 1 Gy/min. Radiofrequency field and X-ray exposures were carried out at room temperature (20°C).
Figure 1: (a) Photograph of the EMEM Devices radiofrequency field (RF) exposure system. (b) Schematic showing the top and bottom cell culture planes of a 2 x 2 x 6 flask matrix. Plasma ray tube centred horizontally above the cell culture flasks. Induced magnetic field (B) and electric field in the culture medium (E) are parallel to the length and width of the flasks, respectively [14].
No cytotoxicity was apparent when cell cultures were exposed to radiofrequency field alone. It was found that the radiosensitising effect of the 1000 Hz-modulated field was more prominent than that of the 100 Hz-modulated field when the interval between X-ray and RF exposures was 4 h [14]. Pre-exposure of the "apparently normal" V79 cells to the 100 and 1000 Hz-modulated fields, 2 h prior X-ray irradiation did not affect the sensitivity of the cells to X-rays. However, the malignant MeWo cells were respectively protected and sensitised by the 100 and 1000 Hz-modulated fields at all time points. On the other hand, the 1000 Hz-modulated field protected the fibroblasts when RF exposure was performed 1 h prior to X-ray irradiation. These findings suggest that RF modulation of cellular radiosensitivity is not only frequency dependent but also reliant on the sequence in which the exposures occur. Also, the observation that the different cell lines do not respond in a similar fashion to the different frequencies may be may be attributed to the notion of the existence cell-specific resonant frequencies at which cells could be rendered sensitive to the X-ray insult [15]. To further investigate the potential of radiofrequency fields in radiotherapy dose reduction, cell survival following an acute (3 Gy), a split dose (1.5 Gy + 1.5 Gy) of X-rays, and 2 Gy given with radiofrequency fields were compared. The key findings are highlighted in Fig. 2 [16]. Cell exposure to RF modulated at 1000 Hz 6 h prior to treatment with 2 Gy of X-rays sensitises the malignant melanoma cells (MeWo) and protects "apparently normal" fibroblasts from the effects of ionising radiation. When compared with 3-Gy treatments, this suggests that use of a radiofrequency field modulated at 1000 Hz can assist in reducing radiation dose by up to 50% without compromising therapeutic benefit. The radioprotective and radiosensitising properties of the RF field seen here corroborate the finding of our earlier study [14].
Figure 2: Surviving fractions for V79 and MeWo cells following various treatment protocols: acute dose of 3 Gy of X-rays, RF exposure alone, and split dose of X-rays (1.5 Gy/fraction) or combination of RF exposure
and 2 Gy of X-rays given 6 h prior to or after each other. Surviving fractions of untreated cell cultures (dashed lines), arrowheads (split dose survival with which survival after combination treatments were compared), pink ovals are for easy comparison between the 'apparently normal" and malignant cells [16].
In a sequel of studies intended to elucidate possible mechanisms underlying the observed modulation of radiosensitivity by the low frequency RF used here, an extended of four human cell lines (melanomas: MeWo -p53 mutant and Be11 - p53 wild-type; prostate carcinoma: DU145 - p53 mutant; normal lung fibroblasts - p53 wild-type) were used [17-21]. Due to a breakdown of the EMEM radiofrequency generation system [14] and our inability to make contact with the supplier (Teli Enterprises LLC), RF exposure for these studies was performed using a PERL M+ oscillator amplifier (Resonant Light Technology Inc., Courtenay, Canada; Serial # PM 171116) [17,18; Fig. 3]. A ProGen II frequency generator (Serial No. PG 171211) was used to generate the modulating frequencies. In this set-up, the estimate induced magnetic flux densities ranged from 6.74 to 22.43 I^T.
29-cm plasma ray tube
Figure 3: (a) Photograph of the Resonant Light Technology radiofrequency field (RF) exposure system, with the PERL M inverted on a cut Styrofoam box. (b) Schematic showing the top and bottom cell culture planes of a 2 x 2 x 4 flask matrix. Plasma ray tube centred horizontally above the cell culture flasks. Induced magnetic field (B) and electric field in the culture medium (E) are parallel to the length and width of the flasks, respectively [17,18].
Factors that were investigated for their potential role in radiosensitivity modulation by radiofrequency fields were: (a) p53 status, (b) DNA damage processing and proliferative activity, (c) metabolic activity, and (d) induction of reactive oxygen species (ROS). The modulation of radiosensitivity by radiofrequency fields that was previously demonstrated was confirmed. In the following, the findings on the potential mechanisms underlying RF-mediated alterations of radiosensitivity in vitro are summarised:
p53 status: The effect of a 27.125 MHz field modulated at 100, 1000, 2000, and 4000 Hz on the radiosensitivity of the four human cell lines (MeWo, Be11, DU145, and L132) was assessed on the basis of colony forming capacity. The wide range of RF-induced magnetic flux density for the different radiofrequency generation systems did not seem to significantly affect RF-modulation of radiosensitivity [14,17,18]. The modulation cellular radiosensitivity by radiofrequency fields was confirmed and appeared to be frequency- and cell-type-dependent [17]. Extending the modulating frequencies to include 2000 and 4000 Hz showed that the extent of RF-mediated radiosensitisation does not steadily increase with increasing the modulating frequency. The demonstrated frequency and cell type-dependence of radiofrequency field modulation of cellular sensitivity to X-ray treatment is consistent with the finding of the earlier studies [14,16]. In the panel of cell lines used here, the p53 wild-type cells showed the highest radiosensitisation and which peaked at a modulating frequency of 1000 Hz, especially when cells were irradiated to 6 Gy [18]. This is consistent with the earlier observation that the sensitisation of the "apparently normal" V79 cells was minimal [14]. These rodent cells possess a nonfunctional and mutated p53 [22]. This finding shows that a radiofrequency field of 27.125 MHz modulated at 1000 Hz is more efficient in modulating large fractional doses of ionising radiation and may be beneficial in
hypofractionated radiotherapy where large doses are administered fraction. Specifically, this could be the case for patients with tumours exhibiting low alpha/beta ratios, like those presenting with prostate cancer. Expanding the panel of cell lines may help in confirming the potential role of low frequency radio waves and cellular gene profiles in the use of radiofrequency fields as radiation protectors and sensitisers. The role of p53 status in the radiofrequency modulation of cellular sensitivity to ionising radiation is also warrants further investigation.
DNA damage processing and proliferative activity: Cellular DNA processing was evaluated using the cytokinesis-blocked micronucleus formation assay. The yield of binucleated cell in the cytokinesis-blocked cultures was used as an indicator of proliferation. The yields of micronuclei (marker of DNA damage) and binucleated cells (marker of proliferation) were correlated with the cellular radiosensitisation that was conferred by 100- and 1000-Hz modulated radiofrequency fields [19]. Binucleation can be used represent cell proliferation, as an elevated binucleation index is indicative of a large proportion of actively progressing cells through the cell cycle [23-25]. An elevated micronucleus yield was linked to a higher degree of radiosensitisation by radiofrequency fields. This correlation was independent of modulating frequency. While an increased proliferative index was correlated with a marked radiosensitisation by the 100-Hz modulated field, a raised proliferative capacity was linked to a slight radiosensitisation when cells were exposed to the 1000-Hz modulated field. From these findings, it can be inferred that that radiofrequency fields interfere with the capacity of cells to process ionising radiation-induced DNA damage and their ability to progress in the cell cycle. Interaction between ionising radiation and radiofrequency fields may have significant ramifications in radiation protection, radiotherapy, and wound healing following preoperative radiotherapy. Although fibroblast proliferation is a precursor of good wound healing, enhanced proliferation in irradiated fibroblasts does not always promote efficient wound healing [26]. Therefore, further validation of these findings in a large panel of cell lines and preclinical systems is of merit.
Metabolic activity: Changes in the metabolic activity of the cell lines was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell lines that displayed larger reductions in metabolic activity emerged more radiosensitised by radiofrequency exposure, relative to when they were treated with radiation alone [20]. This was independent of whether RF exposure occurred prior to or after X-ray irradiation. The finding that metabolic activity in surviving cells remained raised following a combined treatment of X-rays and radiofrequency fields for up to 18 h suggests that radiofrequency fields trigger alterations in metabolic activity to support DNA repair and subsequent cell survival. Changes in cellular metabolic activity in X-ray treated cells following radiofrequency exposure might have significant implications for cellular responses to ionising radiation in the ambience of radiofrequency fields. The sustained metabolic activity observed in X-ray treated cells concomitantly exposed to the radiofrequency may be attributed to the RF fields inducing dormant cells to enter and progress through the cell cycle. This could explain the enhance cell proliferation eluded to earlier [19]. An enhanced proliferation is a reflection of increased G2/M activity [27,28]. As cells G2/M are more radiosensitive would be more susceptible to the radiation insult, thus giving rise to elevated radiosensitisation. This phenomenon requires further elucidation in a large panel of cell lines and preclinical models, if radiofrequency fields of the kind studied here are to be routinely used in radiotherapy as adjuvants.
Induction of reactive oxygen species (ROS): ROS expression was assessed by measurement of cytosolic superoxide dismutase activity in the cell lines following the various treatments. On average, radiation-induced ROS activity was consistently increased by radiofrequency field exposure in all cell lines. However, the degree to which RF exposure enhanced ROS activity did not correlate with the level of radiosensitisation [21]. The finding that ROS activity is elevated by radiofrequency field exposure supports other studies that have demonstrated that electromagnetic fields have a strong influence ROS production in cellular systems [29-31]. The absence of clear link between RF-induction of ROS radiosensitivity is not entirely unexpected as reactive oxygen species are known to play multiple and opposing roles in biological systems. ROS can initiate oxidative stress that leads to cell death or promote malignant cancer acclimatisation to low oxygen tension, cell propagation, and survival [32-34]. Similar studies on an expanded panel of cell lines and preclinical systems are required in order to confirm the potential role of reactive oxygen species in radiofrequency-mediated radiosensitisation.
Acknowledgements
These finding are based on research supported by the National Research Foundation of South Africa (Grant Nos.: 85703, 92741, 100157, 107703) and funding from the Faculty of Medicine and Health Sciences (Stellenbosch University), the Harry Crossley Foundation, and the Cancer Association of South Africa.
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