Constructal approach to cell membranes transport: Amending the ‘Norton-Simon’ hypothesis for cancer treatment.
- 1Dipartimento Energia, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.
- 2Department of Medical Sciences, University of Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy.
- 3Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
- 4ThinkMotu LLC, Wellesley, MA 02481, USA.
To investigate biosystems, we propose a new thermodynamic concept that analyses ion, mass and energy flows across the cell membrane. This paradigm-shifting approach has a wide applicability to medically relevant topics including advancing cancer treatment. To support this claim, we revisit ‘Norton-Simon’ and evolving it from an already important anti-cancer hypothesis to a thermodynamic theorem in medicine. We confirm that an increase in proliferation and a reduction in apoptosis trigger a maximum of ATP consumption by the tumor cell. Moreover, we find that positive, membrane-crossing ions lead to a decrease in the energy used by the tumor, supporting the notion of their growth inhibitory effect while negative ions apparently increase the cancer’s consumption of energy hence reflecting a growth promoting impact. Our results not only represent a thermodynamic proof of the original Norton-Simon hypothesis but, more concretely, they also advance the clinically intriguing and experimentally testable, diagnostic hypothesis that observing an increase in negative ions inside a cell in vitro, and inside a diseased tissue in vivo, may indicate growth or recurrence of a tumor. We conclude with providing theoretical evidence that applying electromagnetic field therapy early on in the treatment cycle may maximize its anti-cancer efficacy.
Nanosecond pulsed electric field inhibits proliferation and induces apoptosis in human osteosarcoma
Recent studies suggest that nanosecond pulsed electric field (nsPEF) is a novel minimal invasive and non-thermal ablation method that can induce apoptosis in different solid tumors. But the efficacy of nsPEF on bone-related tumors or bone metastasis is kept unknown. The current study investigates antitumor effect of nsPEF on osteosarcoma MG-63 cells in vitro.
MG-63 cells were treated with nsPEF with different electric field strengths (0, 10, 20, 30, 40, and 50 kV/cm) and different pulse numbers (0, 6, 12, 18, 24, and 30 pulses). The inhibitory effect of nsPEF on the growth of MG-63 cells was measured by Cell Counting Kit-8 (CCK-8) assay at different time points (0, 3, 12, 24, and 48 h post nsPEF treatment). The apoptosis was analyzed by Hoechst stain, in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and flow cytometric analysis. The expression of osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), and tumor necrosis factor a (TNF-a) was examined by reverse-transcription polymerase chain reaction (RT-PCR) and western blot.
The CCK-8 assay showed that nsPEF induced a distinct electric field strength- and pulse number-dependent reduction of cell proliferation. For treatment parameter optimizing, the condition 40 kV/cm and 30 pulses at 24 h post nsPEF achieved the most significant apoptotic induction rate. Hoechst, TUNEL, and flow cytometric analysis showed that the cell apoptosis was induced and cells were arrested in the G0/G1 phase. PCR and western blot analysis demonstrated that nsPEF up-regulated OPG expression had no effect on RANKL, increased OPG/RANKL ratio.
NsPEF inhibits osteosarcoma growth, induces apoptosis, and affects bone metabolism by up-regulating OPG, indicating nsPEF-induced apoptosis in osteosarcoma MG-63 cells. NsPEF has potential to treat osteosarcoma or bone metastasis. When nsPEF is applied on metastatic bone tumors, it might be beneficial by inducing osteoblastic differentiation without cancer proliferation. In the future, nsPEF might be one of the treatments of metastatic bone tumor.
Osteosarcoma is a malignant bone tumor with high occurrence in children and young adolescents. Retrospective review showed that in the past 30 years, osteosarcoma had a poor prognosis and there was no significant improvement of disease-free survival and the stagnated situation has not improved even with the aggressive use of neoadjuvant chemotherapy and radiation therapy . Patients did not benefit from overtreatment, and as a result, a high rate of lung metastasis, recurrence, and pathological fracture frequently occur, keeping osteosarcoma still one of the lowest survival rates in pediatric cancers . Thus, new therapeutic strategy needs to be developed.
Nanosecond pulsed electric field (nsPEF) is an innovative electric ablation method based on high-voltage power technology, which came into medical application in the last decade . NsPEF accumulates the electric field energy slowly and releases it into the tumor in ultra-short nanosecond pulses, altering electrical conductivity and permeability of the cell membrane, causing both cell apoptosis and immune reaction .Quite different from any other traditional local ablation method, nsPEF accumulate less Joule heating and showed no hyperthermic effects , indicating unique advantage over other thermal therapies such as radiofrequency, cryoablation, microwave, and interstitial laser; nsPEF can be used alone and so avoid the side effect caused by chemotherapy or percutaneous ethanol injection .
We have used nsPEF to ablate tumor and showed the equal outcome as the radical resection with proper indication . Clinical trials and pre-clinical studies from different groups proved that nsPEF has direct antitumor effects by inhibiting proliferation and causing apoptosis in human basal cell carcinoma [8, 9], cutaneous papilloma, squamous cell carcinoma , melanoma [11, 12], hepatocellular tumor , pancreatic tumor , colon tumor [15, 16], breast cancer [17, 18], salivary adenoid cystic carcinoma , oral squamous cell carcinoma , et al. Local ablation with nsPEF indicates the noticeable advantage of not only eliminating original tumors but also inducing an immune reaction, e.g., enhance macrophage  and T cell infiltration  and induce an immune-protective effect against recurrences of the same cancer . The characteristic of electric field on bone metabolism  is extremely helpful for osteosarcoma patients with pathological fracture which leads to poor prognosis [25, 26].
Considering osteosarcoma is especially prevalent in children and young adults during quick osteoblastic differentiation [1, 2], unstable RB gene and p53 gene are commonly involved in this malignant transformation process ; we hypothesize that nsPEF affects osteosarcoma growth by targeting the Wnt/?-catenin signaling pathway, a key signaling cascade involved in osteosarcoma pathogenesis. Here, we investigate nsPEF-induced changes on human osteosarcoma MG-63 cells to determine (1) the dose-effect relationship and time-effect relationship of nsPEF on osteosarcoma cell growth and apoptosis induction and (2) the nsPEF effect on the osteosarcoma cell; osteoblast specific gene and protein expression (receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG)) were measured along with the production of the pro-inflammatory cytokine tumor necrosis factor a (TNF-a).
Materials and methods
Cell lines and cell culture
MG-63 human osteosarcoma cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Sigma, Aldrich, St. Louis, MO, USA). Cells were kept in a humidified atmosphere of 5 % CO2 at 37 °C.
The nsPEF treatment and dose-effect exam
The nsPEF treatment system was made by Leibniz Institute for Plasma Science and Technology, Germany, and an nsPEF generator with duration of 100 ns was applied. Varied electric fields were released in a cell treatment system from 10 to 60 kV/cm. Waveforms were monitored with a digital phosphor oscilloscope (DPO4054, Tektronix, USA) equipped with a high voltage probe (P6015A, Tektronix, USA). MG-63 human osteosarcoma cells were harvested with trypsin and resuspended in fresh DMEM with 10 % FBS to a concentration of 5.0 × 106 cells/mL. Five hundred microliters of cell suspension were placed into a sterile electroporation cuvette (Bio-Rad, US, 0.1-cm gap). Cells were exposed to 100 pulses at 0, 10, 20, 30, 40, 50, and 60 kV/cm electric field strengths, respectively. Under the 50 kV/cm electric field strength, the different pulse numbers were applied (0, 6, 12, 18, 24, and 30 pulses). The experiments were repeated for three times. After incubation for 24 h, cells were calculated by Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan).
Measurement of apoptosis with TUNEL assay, Hoechst stain, and flow cytometry
At different hours after nsPEF treatment (40 kV/cm, 30 pulses), the treated cells were incubated for 0, 3, 12, 24, and 48 h to determine single-cell apoptosis using the assay of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) with In Situ Cell Death Detection Kit (Millipore, USA) and Hoechst stain kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction, as previously described . Under different electric field strengths and with different pulses, the treated cells were incubated for 24 h to detect cell apoptosis by Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). The cell cycle was also analyzed as previously described .
Reverse-transcription polymerase chain reaction
Reverse-transcription polymerase chain reaction (RT-PCR) was performed for assessing the expression of OPG, RANKL, and TNF-a. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house keeping gene, was used as the internal control to calculate the comparative expression. Total RNA was extracted using TRIzol reagent (Sangon, Shanghai, China). The first strand cDNA synthesis from 1 mg of RNA was performed using SuperScript II Reverse Transcriptase (Invitrogen) and Oligo dT primer (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PCR was performed using the oligunucleotides listed as the following. The specific primers were made by Sangon, Shanghai, China, which were listed as the following: RANK: F: CAGGAGACCTAGCTACAGA, R: CAAGGTCAAGAGCATGGA, 95 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min; OPG (264 bp): F: AGTGGGAGCAGAAGACAT, R: TGGA CCTGGTTACCTATC, 95 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min; TNF-a: F: GTGGCAGTCTCAAACTGA, R: TATGGAAAGGGGCACTGA, 94 °C, 40 s; 55 °C, 40 s; 72 °C, 40 s; GAPDH: F: CAG CGACACCCACTCCTC, R: TGAGGTCCA CCACCCTGT, 94 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min.
Western blotting analysis
MG-63 cells (5 × 105) were plated and treated with different doses of nsPEF. Cells were then lysed with a lysis buffer and then quantified. The equal amounts of protein were loaded, and electrophoresis was applied on a 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis mini-gel. Proteins were transferred to a PVDF membrane and blocked with casein PBS and 0.05 % Tween-20 for 1 h at room temperature. Membranes were incubated with mouse monoclonal OPG, anti-OPG (1:500), RANKL (1:200), TNF-a (1:300), GAPDH (1:1000) antibodies which were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibody was purchased from Zhongshan (Zhongshan Golden Bridge, Beijing, China.). The protein expression was visualized with enhanced chemiluminescence reagent (ECL kit, Amersham, UK).
Statistical significance was determined using Student’s t test, using SPSS 13.0. P < 0.05 was considered to indicate a statistically significant result.
NsPEF parameter optimizing by CCK-8 and flow cytometry
CCK-8 assay was used to calculate the IC50 values, and flow cytometry was used to detect apoptosis. There were significant growth inhibition and apoptosis induction in a dose-dependent manner following nsPEF treatment for 24 h. MG-63 cell growth was inhibited in an electric field strength- and pulse number-dependent manner. There was significant (P > 0.001) growth inhibition when electric field strength was 40–50 kV/cm (Fig. 1a) and when pulse number was 30 (Fig. 1d) vs control. Cells were treated by nsPEF and then incubated for 24 h. Apoptotic and dead cells were analyzed by flow cytometry using dual staining with propidium iodide (PI) and Annexin V-FITC. NsPEF induced viable apoptotic cells stained with Annexin. The apoptotic cell rate is significantly increased when electric field strength was 40–50 kV/cm (Fig. 1b, c) and when pulse number was 30 (Fig. 1e, f).
Apoptosis induction at different times post nsPEF treatment
To determine the effects of nsPEF on the induction of apoptosis in MG-63 cells, the Annexin V assay was performed. After 40 kV/cm and 30 pulses of nsPEF treatment, the control and treated cells were stained with Hoechst 33528 (Fig. 2a upper lane) and TUNEL (Fig. 2a lower lane). The statistical analysis of the positive apoptotic cells were counted and shown in Fig. 2b at different hours (0, 3, 12, 24, and 48 h). Apoptotic cells induced by nsPEF treatment were recognized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), detecting DNA fragmentation by labeling the terminal end of nucleic acids. The number or percentages of apoptotic cells detected following nsPEF treatment was shown in Fig. 2b. The quantitative analysis showed the percentages of apoptotic cells detected following nsPEF treatment which were 2.6 % (0 h), 8.8 % (3 h), 21 % (12 h), 42 % (24 h), and 15 % (48 h) without nsPEF treatment. The apoptotic induction 12 and 24 h post nsPEF treatment showed significance (P = 0.01243, 0.00081, respectively, vs control). The cell cycle was analyzed by flow cytometry (Fig. 2c) and statistically analyzed in Fig. 2d, which indicates that nsPEF arrest cells in the G0/G1 phase (Fig. 2d).
The effect of nsPEF on OPG/RANKL, TNF-? gene, and protein expression
With 30 pulses, 24 h post treatment, PCR and western blot were used to determine the different electric field strengths on cell OPG/RANKL, TNF-? gene (Fig. 3a), and the corresponding protein expression (Fig. 3b). NsPEF significantly increased OPG transcription and protein expression at 20–50 kV/cm (Fig. 3a, c). RANKL was almost undetectable both in the control and nsPEF-treated MG-63 cells (Fig. 3a, c). NsPEF slightly down-regulated TNF-a (Fig. 3a, c). The OPG is important in the regulation of bone formation. PCR results showed that the nsPEF-treated cells demonstrated a significantly up-regulation of OPG transcription. Western blot analysis confirmed that nsPEF stimulated osteoprotegerin protein production in the MG-63 cells.
The primary bone malignancy osteosarcoma is still a challenge for orthopedics. For patients who are not suitable for radical resection, the minimal invasive ablation techniques can be used as an alternative to surgery. NsPEF has been proved to be a novel non-thermal ablation method which can activate a protection immune response [21–23]. According to the Clinical Practice Guidelines in Oncology of the National Comprehensive Cancer Network (NCCN), local ablation can be used for curative or palliative intent, either alone or in combination with immunotherapy or chemotherapy . The effect of systemic chemotherapy may be enhanced by the physiological changes produced by ablation . Furthermore, ablation can sometimes be used as a complement to surgery .
A number of studies have demonstrated that local ablation is effective in osteosarcoma [28–30]. To our best knowledge, the application of nsPEF in osteosarcoma has never been reported. The bone-related tumor study is extremely important because many solid tumors tend to have metastasis in bones. The present study applies a new ablation methodology in osteosarcoma and identifies its molecular target. Our data suggest that nsPEF had direct effects on osteosarcoma cells, including the inhibition of tumor cell proliferation and induction of apoptosis. These results are consistent with previous reports. NsPEF inhibits cell proliferation and induces apoptosis in tumor cells [11, 16].
The development of osteoclasts is controlled by cytokine synthesized by osteoblasts like receptor activator of NF-?B ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor ? (TNF-a) .The extension of the current study is the investigation of nsPEF’s effect on bone resorption when nsPEF is in its ablation dosage. OPG is a member of the tumor necrosis factor receptor family. It has multiple biological functions such as regulation of bone turnover. OPG can block the interaction between RANKL and the RANK receptor . NsPEF increased OPG expression in MG-63 in in vitro assays. Our data indicate that nsPEF up-regulated the OPG expression. Bone remodeling can be assessed by the relative ratio of OPG to RANKL . NsPEF had no effect on RANKL expression. Defined as a potent bone-resorbing factor, TNF-a is responsible for stimulating bone resorption. TNF-? exerts its osteoclastogenic effect by activating NF-?B with RANKL . Our results show that in osteosarcoma MG-63, in addition to apoptosis induction, nsPEF can regulate bone metabolism through adjusting OPG/RANKL ratio.
TNF-a expression still needs further investigation due to the weak expression. But, it is the key cytokine that we assume which would change the local inflammatory microenvironment in the ablation zone.
The limit of the current study
In this in vitro study, the MG-63 osteosarcoma cell line is used as a model system. Therefore, results obtained from cultured cells only gave hints for the nsPEF treatment of osteosarcoma. The current results need to be tested in an in vivo osteosarcoma model, e.g., MG-63 cell xenografts.
NsPEF can be considered as a potential therapeutic intervention to suppress bone remodeling and osteoclast activity involved in osteosarcoma. Further in vivo studies are required to optimize the dosing regimen of nsPEF to fully study its antitumor potential in the bone microenvironment.
All authors acknowledge Dr.Karl H. Shoenbach, Dr. Stephen Beebe, and Mr. Frank Reidy from Old Dominion University for their kind support.
This research is supported by National Natural Science Foundation of China (Nos. 81372425 and 81371658), National S & T Major Project (No. 2012ZX10002017), Zhejiang Natural Science Foundation (LY13H180003), and Xinjiang Cooperation Project (2014KL002).
Xudong Miao and Shengyong Yin contributed equally to this work.
The authors declare that they have no competing interests.
XM and SY carried out the molecular genetic studies and drafted the manuscript. ZS carried out the immunoassays. YZ participated in the design of the study and performed the statistical analysis. XC conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.
Low Intensity and Frequency Pulsed Electromagnetic Fields Selectively Impair Breast Cancer Cell Viability
A common drawback of many anticancer therapies is non-specificity in action of killing. We investigated the potential of ultra-low intensity and frequency pulsed electromagnetic fields (PEMFs) to kill breast cancer cells. Our criteria to accept this technology as a potentially valid therapeutic approach were: 1) cytotoxicity to breast cancer cells and; 2) that the designed fields proved innocuous to healthy cell classes that would be exposed to the PEMFs during clinical treatment.
MCF7 breast cancer cells and their normal counterparts, MCF10 cells, were exposed to PEMFs and cytotoxic indices measured in order to design PEMF paradigms that best kill breast cancer cells. The PEMF parameters tested were: 1) frequencies ranging from 20 to 50 Hz; 2) intensities ranging from 2 mT to 5 mT and; 3) exposure durations ranging from 30 to 90 minutes per day for up to three days to determine the optimum parameters for selective cancer cell killing.
We observed a discrete window of vulnerability of MCF7 cells to PEMFs of 20 Hz frequency, 3 mT magnitude and exposure duration of 60 minutes per day. The cell damage accrued in response to PEMFs increased with time and gained significance after three days of consecutive daily exposure. By contrast, the PEMFs parameters determined to be most cytotoxic to breast cancer MCF-7 cells were not damaging to normal MCF-10 cells.
Based on our data it appears that PEMF-based anticancer strategies may represent a new therapeutic approach to treat breast cancer without affecting normal tissues in a manner that is non-invasive and can be potentially combined with existing anti-cancer treatments.:
There is a growing interest in the use of electromagnetic fields as an anticancer treatment –. The search for new therapeutic strategies is particularly active in the field of oncology where standard antineoplastic treatments, based on chemotherapeutic drugs and/or radiotherapy, possess potentially detrimental secondary effects and on their own often fall short of providing a complete and resilient recovery. Fueling this recent interest is the fact that extremely low-frequency and low-intensity pulsed electromagnetic fields (PEMFs) have been shown to be innocuous, possibly even beneficial , –, to normal cell types. On the other hand, certain malignant cell classes have been shown to be particularly vulnerable to their effects , –. A potential value of extremely low frequency PEMFs hence lies in their use as an adjuvant treatment to more traditional chemo- and radiotherapies with the aim of reducing their dosage, mitigating any harmful secondary side effects and enhancing patient prognosis. Despite recent successes, however, the types of signals applied and cancer classes tested varied widely, producing a wide range of killing efficiencies and succeeding in forestalling concurrence in this area of research , –. A clear determination of the types of cancer most susceptible to PEMFs and their subsequent optimization for targeted killing will be needed before they can be used to selectively remove cancer cells from a heterogeneous population of malignant and healthy cells.
Here we show that the ability of ultra-low intensity and frequency PEMFs to selectively kill breast cancer cells depends exquisitely on field parameters. MCF-7 breast cancer cells are selectively vulnerable to PEMFs within a discrete window of PEMF signal parameters and times of exposure with resolutions of mTeslas and tens of minutes, respectively. Using five independent means of monitoring cancer cell death we obtained identical findings; selective killing of MCF7 cells was best achieved with PEMFs of 3 mT peak-to-peak magnitude, at a pulse frequency of 20 Hz and duration of exposure of only 60 minutes per day. By stark contrast, this same pulsing paradigm (cytotoxic to MCF-7s) was innocuous to normal MCF-10 breast cells. PEMF-based therapeutic strategies might thus provide a manner to control certain classes of cancer while minimally implicating healthy tissues.
Materials and Methods
Human adenocarcinoma MCF7 cells and human not tumorigenic MCF10 cells were provided by ATCC (Manassas, VA, USA). MCF7 cells were grown in D-MEM (Life Technologies Corporation, Gibco, Paisley, United Kingdom) supplemented with fetal calf serum (10%) (Life Technologies Corporation,Gibco, Paisley, United Kingdom), L-glutamine (1%) (Life Technologies Corporation, Gibco, Paisley, United Kingdom) and penicillin-streptomycin (1%) (Sigma-Aldrich, St. Louis, MO, USA). MCF10 cells were cultured in D-MEM/F12 (Life Technologies Corporation, Gibco, Paisley, United Kingdom) supplemented with fetal calf serum (5%) (Life Technologies Corporation, Gibco, Paisley, United Kingdom), EGF (0.02%) (Peprotech, NJ, USA), hydrocortisone (0.05%) (Sigma-Aldrich, St. Louis, MO, USA), insulin (0.1%) (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin (1%) (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained at 37°C in a standard tissue culture incubator (Vitaris AG, Baar, Switzerland) in an atmosphere of 95% humidity and 5% CO2.
PEMFs exposure system
The PEMF exposure setup, described in Text S1 and illustrated in Figure S1 A-C, was housed inside a standard cell culture incubator (Vitaris AG, Baar, Switzerland) providing a humidified environment at 37°C, but lacking CO2 regulation. The cells were exposed to an asymmetric pulsed magnetic field while continuously monitoring the field strength and temperature. The non-exposed (control) cells were placed within the same incubator for identical periods, but shielded from the magnetic fields by a mu metal enclosure surrounding the coils. Thus, all cells were exposed to the same climate and temperature.
MCF7 and MCF10 cells were seeded in T25 flasks (SPL Life Sciences, Korea) at concentrations of 6.5×105cells/ml and 6.7×105 cells/ml, respectively. After 24 hours of being plated the cells were washed with PBS (Life Technologies Corporation, Gibco, Paisley, United Kingdom), given fresh medium and exposed to PEMFs for the first of three daily trials; media was not changed from this point onward. An asymmetric pulsed magnetic field of 6 ms interval at a repetition rate of 20 and 50 Hz were applied at flux densities of 2.0, 3.0 and 5.0 mT (peak-to-peak) for 1 hour/day for three days. Whereas exposure to PEMFs at a repetition rate of 20 Hz caused a significant increase in cancer cells death that was dependent on PEMF amplitude, PEMFs applied at a repetition rate of 50 Hz did not produce any noticeable effects over cell viability and were not dealt with further in this manuscript (Figure S2 A-B). To test for effects of different exposure durations, cells were exposed to PEMFs of 3 mT magnitude and at a repetition rate of 20 Hz for 30, 60 or 90 minutes per days for one, two or three days. Cells were collected and analyzed on the first, second or third day for analysis, depending on the assay being conducted.
Trypan blue assay
After a given PEMF exposure protocol, cells were detached, spun down at 1200 rcf for 5 min, resuspended in 1 ml of PBS and incubated in trypan blue at 11 (Sigma-Aldrich, St. Louis, MO, USA). A homogeneous suspension of cells was then deposited into a Burker chamber (BRAND GMBH + CO KG, Wertheim Germany), viewed microscopically and counted. The percentage of dead cells was obtained by calculating the ratio of trypan blue positive cells in treated and untreated samples. In some cases cells were allowed to recover for up to 48 hours after their last PEMF exposure. These cells were then detached, stained with trypan blue (Sigma-Aldrich, St. Louis, MO, USA) and the number of dead cells calculated relative to control.
Apoptosis determination by DNA strand break detection
Apoptosis was measured by means of an Apo-direct kit (BD biosciences, Allschwil, Switzerland) that labels DNA strand breaks using FITC-dUTP. After each treatment 5×105cells were collected and then fixed and stained accordingly to the manufacturer’s instructions. The assay was run on a FACS Calibur (BD Biosciences, Allschwil, Switzerland) flow cytometer using the positive and negative controls provided in the kit as well as an additional positive (death) control given by exposing MCF7 or MCF10 cells to 1 mM H2O2 overnight. H2O2 applied in this manner resulted in 87% ± 2% (+/– SD, n=4) and 82% ± 3% (+/– SD, n=4) lethality in MCF7 and MCF10 cells, respectively. The FITC fluorescence (520 nm) was detected in the FL1 channel and quantifies the amount of DNA strand breaks. For each measurement, 20,000 cells were acquired and analyzed by Flow Jo software (vers. 7.6.5) (Tree Star Inc. ON, USA).
Analysis of cellular electrical properties by means of Impedance microflow cytometer
Impedance flow cytometry (IFC) was conducted on a prototype provided by Amphasys AG (Root Längenbold (LU), Switzerland). Concisely, the apparatus consists of a microfluidic chip, outfitted with a pair of microelectrodes that measure changes of electrical impedance as cells pass through dual interrogation points in response to an alternating current at four user-defined frequencies in the mid frequency (MF) and high frequency (HF) bands –. The obtained data (amplitude, phase and cell velocity) were automatically converted into a standard FCS3 format and analyzed with Flow Jo (vers. 7.6.5) (Tree Star Inc. ON, USA).
After treatment cells were collected, resuspended in PBS at a concentration of 4–5×106 cells/ml and pumped through the chip at a maximum velocity of 1 cm per second, 500–1000 cells per second. For each measurement, 20,000 cells were analyzed at a frequency of 0.5 MHz to monitor apoptosis –,  or 9 MHz to determine metabolic status –, –. Each sample was run in parallel with polystyrene beads (8 µm) (Sigma-Aldrich, St. Louis, MO, USA) to obtain a standard signal response over the entire frequency spectrum, establishing a set point.
Apoptosis determination by Annexin V staining
An Annexin V/Propidium iodide (BD biosciences, Allschwil, Switzerland) assay was used to monitor the progression of apoptosis at distinct stages. Monitoring the dual staining pattern of Annexin V (FITC- conjugated) and propidium iodide (PI) allowed for the identification of early (Annexin V + and PI -) and late apoptosis as well as cells having undergone necrosis (dead cells, Annexin V and PI +). After each treatment, 3×105cells were collected and stained as specified by the manufacturer’s instructions. Staining was assayed on a FACS Calibur (BD Biosciences, Allschwil, Switzerland), recording 20,000 cells for each measurement. Fluorescence was detected in the FL1 and FL2 channels for FITC (Annexin V) and PI, respectively. Data were acquired and analyzed by Flow Jo software (vers. 7.6.5) (Tree Star Inc. ON, USA).
All histogram data were presented as mean ± SD (standard deviation) of at least 3 independent experimental runs (range=3 to 5) consisting of between 1 to 3 replicates for each biological parameter analyzed. The exact number of measurements in each presented data point is reported in the figure legend and is indicated in brackets (n). Statistics were performed using the Wilcoxon Rank-Sum Test (two-tailed) comparing each treated sample to relative control (sham-exposed sample) for all the cell lines used. A p-value <0.05 was considered statistically significant (*) and a p-value < 0.005 was considered highly significant (**).
PEMFs increase breast cancer cell death as detected by Trypan Blue inclusion
Our objective was to devise a set of treatment protocols that could potentially translate into the clinical arena to slow cancer growth, while proving harmless to healthy tissues. We focused on a breast cancer cell model given our previous success using PEMFs to slow their growth . To ascertain the sensitivity of normal and cancer cells to PEMFs we exposed MCF7 breast cancer cells and their normal breast epithelial counterparts, MCF10s, to PEMFs of magnitudes between 2 mT and 5 mT and at a repetition rate of 20 Hz for 1h per day for three days. Following the last exposure (day 3) all samples were harvested and stained with trypan blue to quantify cell death and compared to otherwise identically treated control (non-exposed) cultures. A highly significant reduction in the percentage of surviving MCF7 cells was observed in response to exposure to 3 mT PEMFs. By contrast, exposure of identical MCF-7 cultures to PEMFs of either 2 mT or 5 mT amplitudes resulted in less significant levels of cell death (Fig 1A). On the other hand, exposure to 3 mT PEMFs, which proved the most cytotoxic to MCF-7 cancer cells, was innocuous to “wild type” MCF10 cells (as were 2 and 5 mT PEMFs) and moreover, appeared to have even accentuated their survival (mitigating resting levels of apoptosis) relative to unexposed cells (also see Figure S5). We next sought to determine the best exposure interval to 3 mT PEMFs to kill breast cancer cells. Figure 1B depicts cell death as a function of duration of exposure to 3 mT PEMFs (20 Hz). Cells were exposed to 3 mT PEMFs for either 30, 60 or 90 minutes per day for 3 days before assaying for cell death. MCF7 cells were most susceptible to PEMF exposures of 60 minutes duration, whereas exposure times 50% shorter (30 minutes) or 50% longer (90 minutes) than this resulted in significantly less amounts of cell killing (Fig 1B). Once again, MCF10 cell viability was not compromised by PEMF exposure of any duration. Indeed, PEMFs appeared to make MCF10 cells more resistant to undergoing apoptosis, particularly in response to the 60-minute exposure regimen that proved most cytotoxic to MCF7 cells (Figure S5). The data thus reveals a discrete set of PEMF parameters (magnitude, frequency and duration of exposure) that are most cytotoxic to breast cancer cells, whereas the identical set of PEMFs parameters were apparently harmless to non-malignant cell types (also seeFigures S3 and S4).
To ascertain whether the PEMFs-induced cytotoxicity reported here is a cumulative response or requires a threshold level of cellular insult to become evident, we treated cells with 3 mT PEMFs for either 60 or 90 minutes per day for 1, 2, or 3 days and next quantified the total number of dead and living cells. Whereas in the unexposed cultures total cell number steadily increased throughout the three days of trial, exposure to 60 or 90 minutes of PEMFs per day either totally abrogated or slowed the increase in cell number, respectively (Fig 2). On the other hand, the absolute number of dead (trypan blue positive) cells did not scale down in proportion to the decrease in total cell number as might be expected if cell proliferation was simply being slowed, but instead, increased. Notably, on the third day, in response to 60 minutes of daily exposure to PEMFs (3 mT), the total number of cells in the culture decreased, whereas the total number of dead cells increased, by –40% (+/–6% (SD); n=12) ((total cells in control sample – total cell in treated sample)/total cells in control sample)) and +20% (+/–13% (SD); n=12) ((dead cells in control sample – dead cell in treated sample)/dead cells in control sample)), respectively, indicating heightened cytotoxicity in response to PEMFs. Figure 3 shows that the increase in cell loss with time is greatest in cultures treated for 60 minutes per day, rather than 90 minutes per day.
Assessment of PEMF-induced apoptosis by detecting DNA strand breaks
Our Flow Cytometric (FCM) determination of apoptosis was assayed with identical PEMF parameters (days of consecutive exposure, durations of exposure, field amplitudes and frequency) as those utilized for trypan blue assessment of killing efficiency with identical results. Figure 4A shows an overlay of MCF7 cells exposed to PEMFs of three distinct intensities (2, 3 or 5 mT) for 60 minutes per day. A shift to the right (greater FL1-H values) of a cell population reflects greater DNA damage. As previously demonstrated, MCF7 cancer cells are particularly vulnerable to 3 mT PEMFs. Figure 4B shows the extent of 3 mT PEMF-induced DNA strand breaks following 30, 60 or 90 minutes exposures per day. Once again, 60 minutes of 3 mT PEMFs for three consecutive days gave the greatest DNA damage in MCF7 cancer cells. And, once again, stronger fields (5 mT) or longer exposures (90 minutes per day) were less cytotoxic to MCF7 cells (Fig 4A-D). Further paralleling our trypan blue results, MCF10 normal breast epithelial cells were not harmed by any of the PEMF paradigms tested, particularly those observed to be especially cytotoxic to MCF7 cells. Indeed, a slight protective effect (a leftward shift to lower FL1-H values) was again discerned in MCF10 cells in response to the PEMF parameters that were most cytotoxic to MCF7 breast cancer cells (Fig 4E; see also Figure S5). To investigate if the previously described increase in DNA fragmentation observed in MCF7 cells after 3 days of PEMF treatment was cumulative with time, we stained cells after 1, 2 or 3 consecutive days of exposure to either 60 or 90 minute of 3 mT PEMFs. Although PEMF-induced DNA damage increased with time, it only really obtained significance from control levels after the third day and was particularly pronounced in response to 60-minute daily exposures (figure 5 A-D). Our FCM analysis thus corroborates and strengthens our trypan blue results, indicating that treatment with 3 mT PEMFs for 60 minutes per day were most effective at killing MCF7 breast cancer cells while leaving healthy cell classes (MCF10) unharmed.
Determination of PEMF-induced apoptosis by Impedance Flow Cytometry
Impedance Flow Cytometry (IFC) assesses real-time cell viability by monitoring cellular electrical properties in behaving cells –, . In the dot plot generated from monitoring the entire cell population’s electrical characteristics at a scan frequency of 0.5 MHz dead cells reside in the far lower left quadrant (low impedance phase and magnitude values). PEMFs produced a shift in MCF7 cells to the lower left quadrant, particularly in response to 3 mT PEMFs, which gave the greatest separation between living (right) and dying (left) cells (Fig 6A). Figure 6B shows the results of MCF7 cells exposed to 3 mT PEMFs for either 30, 60 or 90 minutes per day for three days. In agreement with our previous trypan blue and FCM assessment of apoptosis, cells exposed to 60 minutes of 3 mT PEMFs per day exhibited the greatest percentage of dead cells as detected by IFC (Fig 6 C-D). In stark contrast, yet in further confirmation of our previous results, MCF10 cells were slightly benefitted by these same PEMF parameters (Fig 6E, see also Figure S5).
Assessment of cell metabolic status after PEMF treatment with IFC
At higher scan frequencies the IFC discerns metabolic status –, . At a scan frequency of 9 MHz the IFC detects two populations of cells, the right-most population (higher phase values) reflects cells experiencing the initial stages of metabolic stress –, –. After three days of exposing MCF7 cells to PEMFs the magnitude of right-most population augmented, the greatest right-shift coinciding exactly with those parameters (3 mT, 20 Hz, for 60 min/day for 3 days) producing the greatest cell death in response to PEMFs (Fig 7 A-D). And, once again, MCF10 normal breast cells were apparently benefitted by PEMFs as determined by IFC analysis at 9 MHz (Fig 7 D, see also Figure S5). Due to the relatively broad scope of the phenotype (metabolic stress) the effect is the largest we have measured in response to PEMFs (see next, see also Figure S5).
To independently validate that IFC effectively detects apoptosis and metabolic status in our cell system we treated MCF7 cancer and MCF10 normal cells with 1 mM H2O2 to evoke cell death to an extent of 87% ± 2% (+/– SD, n=4) and 82% ± 3% (+/– SD, n=4), respectively. When analyzed by IFC at a scan frequency of 0.5 MHz cells treated with H2O2 were displaced to the far lower left quadrant (Fig 8A; cf Fig 6A-D). Also, confirming that a cell population undergoing the initial stages of metabolic stress is indeed shifted to the right (in IFC scans at 9 MHz) we obtained an analogous right-shift in MCF7 cells after overnight exposure to 1 mM H2O2 (Fig 8B; cf Fig 7A-D). Hence, IFC does appear to be a viable method to monitor cancer cell viability.
Assessment of PEMF-induced apoptosis by Annexin V staining
To further corroborate our trypan blue, FCM and IFC data demonstrating the induction of apoptosis in MCF7 cancer cells in response to PEMF exposure, we performed Annexin V/PI assays, discriminating cells in early apoptosis (Annexin V+/PI-) from dead and damaged cells (propidium iodide +). MCF7 (cancer) and MCF10 (normal) cells were directly exposed to the PEMFs paradigms we previously found to be most cytotoxic to MCF7 cells, 3 mT for 60 minutes per day. Figure 9A shows that PEMF treatment resulted in a 13% increase in Annexin V+ MCF7 cells relative to control, quantitatively agreeing with our other PEMF-induced cytotoxic assessments assayed with trypan blue (treated – control: 11% dead cells), FCM (treated – control: 14% dead cells), IFC at scan frequency of 0.5 MHz (treated – control: 16% dead cells) and IFC at scan frequency of 9 MHz (treated – control: 25%). As previously demonstrated with all the other apoptosis assays we performed, MCF10 cells were not adversely affected by these same PEMF parameters (Fig 9B) (also see Figure S5).
Motivated by studies demonstrating the safety of very low frequency and intensity PEMFs ,  and extending from our previous work , demonstrating that MCF7 cancer cells are selectively vulnerable to 20 Hz pulsed electromagnetic fields, we investigated the effects of PEMFs on human breast epithelial cells of malignant (MCF7) and non-malignant (MCF10) phenotypes. Cytotoxic sensitivity to certain PEMFs parameters was entirely restricted to the malignant phenotype and exhibited a clear dependency on the duration, frequency and intensity of the PEMFs employed. Specifically, breast cancer cells of the MCF7 lineage were most vulnerable to PEMFs of 3 mT magnitude, at a repetition rate of 20 Hz and for an exposure interval of 60 minutes per day (Fig 1 A-C). These same PEMF parameters, although cytotoxic to MCF7 cells, were slightly protective to non-malignant breast epithelial cells of an identical host lineage, MCF10 (see Figure S5). For these experiments we limited our analysis to within three days of exposure to remain within the realm of a clinically feasible therapeutic strategy. Three days was also chosen as an appropriate end point to our analysis as it avoided the overgrowth of control cells. In a tissue culture paradigm such as ours, staying below cell confluence would minimize the potential contributions of cell density/contact-induced changes in biochemical status or nutrient deprivation to our measured differences. The possibility hence remains, that increasing the number of days of exposure to PEMFs may enhance the specificity and efficiency of cancer cell killing. The design of longer time course experiments will be the focus of our future studies. Nonetheless, our results, although relatively modest are sufficiently provocative (in terms of their reproducibility and selectivity) to merit future studies aimed at further evolving this approach and yet, are consistent with previous studies demonstrating that sensitivity to electromagnetic fields depends on the signal parameters used as well as the type of cells exposed to the fields , , , –.
For this study we focused our attention on PEMF parameters that: 1) could practically translate into the clinical arena with reference to duration of exposure and 2) were innocuous to healthy cell classes collaterally exposed to PEMFs during clinical treatment. Our results are notable given that: 1) our most effective exposure time to induce cancer cell (MCF7) death was only one hour per exposure rather than 3–72 hours as previously reported ,– and; 2) the field paradigms we designed were apparently innocuous to normal cells (MCF10). As of yet, we have not achieved complete “selective” killing with PEMFs. Although this objective might be achieved with further fine-tuning of the PEMF parameters (exposure magnitude, duration, signal shape, number of days of treatment) we cannot then exclude the possibility that other tissues type might then be implicated in the death pool. Quite notable, however, were the diametrically opposed responses of MCF7 (cancer) and MCF10 (normal) cells to PEMFs, widening the cytotoxic gap between exposed cancer and exposed normal cells. Potentially, PEMFs might prove useful as a non-invasive adjuvant treatment to be combined with other common anti-cancer therapies.
The selective killing of cancer cells with PEMFs was corroborated by four independent methodologies using five different analytical paradigms, covering the full gambit of stages leading to ultimate cell death. Firstly, our trypan blue results gave the number of cells in a late stage of cell dying known as “postapoptotic necrosis” or “secondary necrosis” (Fig 1 A-B, 2 A-D and 3 A-B) , –. Secondly, our FCM analysis detected DNA breaks prior to cell death ,  and occurring downstream of calcium-stimulated caspase activation (Fig 4 A-E and 5 A-D) . Thirdly, we investigated the progression of apoptosis using Impedance Flow Cytometry (IFC) that detects changes in the electrical properties of cells reflecting physiological status –, , – at two frequencies: 1) 0.5 MHz, to ascertain the number of cells having undergone apoptosis (Fig 6 A-E) –, and 2) 9 MHz, to monitor changes that coincide with the onset of cellular stress (Fig 7 A-E) –, –. Several recent publications have supported the value of IFC to gauge cell viability –, . Finally, we employed an Annexin V/PI assay to distinguish early apoptotic cells from damaged or already dead cells (Fig 9 A-B) –. In all five assays of cell viability identical PEMF parameters produced the greatest degree of cell damage to MCF-7 breast cancer cells, 3 mT intensity for 60 minutes a day, demonstrating a clear and discrete window of vulnerability of MCF7 cells to PEMFs of given characteristics. Stronger fields, longer exposures, or higher frequencies to these empirically determined values (3 mT, 20 Hz, 60 minutes exposures per day) were less cytotoxic to MCF7 cells, clearly demonstrating the importance of field optimization for the eventual killing of malignant cell classes with PEMFs.
A clear window of vulnerability of cancer cells to PEMFs exists; more is not necessarily better. That weaker fields, or less exposure to them, are less lethal, upon first impression, might seem somewhat intuitive. However, the fact that stronger, or longer, exposure to fields is less efficient at killing, implies some specifically of biological action, rather than a straightforward dose-dependent accumulation of generalized damage over a susceptible cell status. The validity of the described window effect is implicitly substantiated within the context of our data presented herein, the fact that five independent assays (four distinct methodologies) of measuring cell viability gave the identical result and produced similar magnitudes of cell death (also see Figure S5). The cytotoxic-dependency on exposure duration was so robust that it was also apparent when examining the time course in the development of cytotoxicity during three days of consecutive PEMF exposure. That is, 60-minute daily exposures to PEMFs gave greater ratios of cell death (figure 3) and greater amounts of DNA fragmentation (figure 5) than 90 minutes of daily exposure. Moreover, the PEMF parameters that were most cytotoxic to MCF7 breast cancer cells proved most beneficial to MCF10 normal breast cells. Similar window effects have been reported in the field of electromagnetics and have been openly discussed in the literature, yet there are no accepted models to explain their existence , –. Within the Protection Guidelines Report of the International Commission on Non-Ionizing Radiation  it is stated, “Interpretation of several observed biological effects of AM (amplitude modulated) electromagnetic fields is further complicated by the apparent existence of “windows” of response in both the power density and frequency domains. There are no accepted models that adequately explain this phenomenon, which challenges the traditional concept of a monotonic relationship between the field intensity and the severity of the resulting biological effects.”
At this juncture, however, the relative contributions of an actual slowing of cell proliferation and the induction of cell death to the overall effect of PEMFs is unclear (cf figure 2), as is the rate and extent of absorption of dead cells by the culture after their demise. Therefore, although cell cycle withdrawal possibly resulting from PEMFs may contribute to observations reported here, the most directly measurable effect is that of induced apoptosis. Nonetheless, the capacity of PEMFs to slow the proliferation of a cancer cell class also would be positive clinical outcome and of relevance in advancing PEMF-based anti-cancer therapies.
The molecular mechanisms whereby cancerous (MCF7) cells are compromised yet, healthy (MCF10) cells are not fully understood and yet, of utmost importance for the ultimate development of PEMF-based strategies to combat cancer and will be the focus of our future investigations. We speculate that the window effect observed in this study results from changes in intracellular calcium handling in response to PEMF exposure. Calcium signaling is renowned for its multimodal effects relying on intracellular calcium increments that: 1) result from both calcium influx across the cell surface membrane and release from intracellular membrane-delimited compartments; 2) are simultaneously coded in space, time and holding level; 3) exhibit negative- and positive-feedback regulatory mechanisms and; 4) are coordinated by dynamic changes in membrane organization –. As a commonly reported consequence of PEMF exposure is elevations of intracellular calcium level  one possibility is that PEMFs mediate their effects via influencing intracellular calcium signaling pathways. In the context of this report 3 mT PEMFs at a frequency of 20 Hz for 60 minutes per day would create the “correct” combination of calcium signals that would most effectively result in cell death. Indeed, it has been previously shown that chelating or augmenting intracellular calcium accordingly spares or compromises MCF7 survival, respectively –. The shift to the right observed at 9 MHz in IFC (Fig 4 A-D) likely reflects changes in membrane complexity and cytoplasmic reorganization (change in whole-cell capacitance) –, – that coincide with the establishment of cytomorphological features that reflect the modulation of biochemical pathways that, in turn, regulate the delicate balance between cell proliferation and apoptosis including, modifications in mitochondrial metabolism downstream of changes in intracellular calcium levels –, , . Future studies of ours will focus on the effects of PEMFs over cytosolic calcium increments.
Non-malignant MCF10 cells were unaffected, or even fortified, by the PEMF paradigms producing the greatest damage in MCF7 cells, revealing another level of specificity of action and supporting the possibility that it may be ultimately feasible to selectively remove cancer cells from an organism without implicating normal tissues in the death pool using PEMF-based technologies (Figs 1 A-B, ?,4E,4E, ?,6E,6E, ?,7E,7E, ?,9B9B and ). The immunity of MCF10 cells to PEMFs might suggest that their endogenous calcium homeostatic mechanisms are capable of buffering, or even exploiting, small increments in intracellular calcium concentrations, whereas MCF7 cells are not able to withstand even modest perturbations in cytosolic calcium levels, a supposition that is supported by recently published studies–. In further support for such a calcium-dependent mechanism of preferential killing of malignant cells it has been shown that Panaxydol, a derivative of Panax ginseng that induces sustained elevations in cytosolic calcium, preferentially induces apoptosis in cancer cells (including MCF7s) but not normal cells . Such a selective calcium-dependent mechanism of cancer cell killings may eventually help in the refining of PEMF-based technologies to better execute the preferential killing of breast cancer cells in clinical settings.
We would like to acknowledge Dr Malgorzata Kisielow and Ms Anette Schütz of the Flow Cytometry Laboratory of the ETH and University of Zürich for expert technical assistance during the FCM acquisition and analysis. Finally, we would like to thank the Statistical Consulting group of the ETH for their assistance in elaborating our statistical analysis.
This study was partially supported by the Swiss Federal Office of Public Health (http://www.bag.admin.ch/) under the mandate number 11.003272, “Effects of pulsed electromagnetic fields on the proliferation of different mechano-sensitive cell types”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.
Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields.
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk Virginia, 4211 Monarch Way, Norfolk, Virginia 23508, USA.
Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals.
Acc Chem Res. 2012 Apr 30. [Epub ahead of print]
Detecting and Destroying Cancer Cells in More than One Way with Noble Metals and Different Confinement Properties on the Nanoscale.
Laser Dynamics Laboratory, Department of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
Today, 1 in 2 males and 1 in 3 females in the United States will develop cancer at some point during their lifetimes, and 1 in 4 males and 1 in 5 females in the United States will die from the disease. New methods for detection and treatment have dramatically improved cancer care in the United States. However, as improved detection and increasing exposure to carcinogens has led to higher rates of cancer incidence, clinicians and researchers have not balanced that increase with a similar decrease in cancer mortality rates. This mismatch highlights a clear and urgent need for increasingly potent and selective methods with which to detect and treat cancers at their earliest stages. Nanotechnology, the use of materials with structural features ranging from 1 to 100 nm in size, has dramatically altered the design, use, and delivery of cancer diagnostic and therapeutic agents. The unique and newly discovered properties of these structures can enhance the specificities with which biomedical agents are delivered, complementing their efficacy or diminishing unintended side effects. Gold (and silver) nanotechnologies afford a particularly unique set of physiological and optical properties which can be leveraged in applications ranging from in vitro/vivo therapeutics and drug delivery to imaging and diagnostics, surgical guidance, and treatment monitoring. Nanoscale diagnostic and therapeutic agents have been in use since the development of micellar nanocarriers and polymer-drug nanoconjugates in the mid-1950s, liposomes by Bangham and Watkins in the mid-1960s, and the introduction of polymeric nanoparticles by Langer and Folkman in 1976. Since then, nanoscale constructs such as dendrimers, protein nanoconjugates, and inorganic nanoparticles have been developed for the systemic delivery of agents to specific disease sites. Today, more than 20 FDA-approved diagnostic or therapeutic nanotechnologies are in clinical use with roughly 250 others in clinical development. The global market for nano-enabled medical technologies is expected to grow to $70-160 billion by 2015, rivaling the current market share of biologics worldwide. In this Account, we explore the emerging applications of noble metal nanotechnologies in cancer diagnostics and therapeutics carried out by our group and by others. Many of the novel biomedical properties associated with gold and silver nanoparticles arise from confinement effects: (i) the confinement of photons within the particle which can lead to dramatic electromagnetic scattering and absorption (useful in sensing and heating applications, respectively); (ii) the confinement of molecules around the nanoparticle (useful in drug delivery); and (iii) the cellular/subcellular confinement of particles within malignant cells (such as selective, nuclear-targeted cytotoxic DNA damage by gold nanoparticles). We then describe how these confinement effects relate to specific aspects of diagnosis and treatment such as (i) laser photothermal therapy, optical scattering microscopy, and spectroscopic detection, (ii) drug targeting and delivery, and (iii) the ability of these structures to act as intrinsic therapeutic agents which can selectively perturb/inhibit cellular functions such as division. We intend to provide the reader with a unique physical and chemical perspective on both the design and application of these technologies in cancer diagnostics and therapeutics. We also suggest a framework for approaching future research in the field.
Cell Biol Int. 2002;26(7):599-603.
Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.
Biomedical Physics Unit, Department of Physics, Wuhan University, Wuhan, 430072, China.
Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.
Technol Health Care. 2011;19(6):455-67.
Solid Ehrlich tumor growth treatment by magnetic waves.
Department of Biophysics, Faculty of Science, Cairo University, Giza, Egypt.
In this work the retardation of Ehrlich tumor growth implanted in mice was studied by employing 4.5 Hz magnetic field. Eighty female Balb/c mice were used, twenty as normal group; the other sixty mice were inoculated with Ehrlich tumor, then they were divided equally into three groups namely A, B and C. Group A (control group) animals were not exposed to the magnetic field. The tumors in the thigh of the animals of group B were exposed to 4.5 Hz, 2 Gauss square wave magnetic field by using a small solenoid connected to a power square wave generator. Group C animals were whole body exposed inside a large solenoid to 4.5 Hz, 2 Gauss square wave magnetic field. Both groups B and C were exposed for a period of 2 weeks at a rate 2 hours per day. Tumor volume, survival period, histological examination and dielectric relaxation of the tumor were measured to investigate the activity of the tumor of the exposed and the unexposed animals. The results indicated that exposing the tumor tissue to 4.5 Hz square wave magnetic field for 2 weeks at a rate 2 hours/day inhibited tumor growth and increased the survival period of the animals. However, group B showed more improvements than did group C. This was attributed to some distortions in the square waveform in the large solenoid (group C). By comparing data from current and previous work, it was concluded that the use of magnetic waves showed better results over previously published work using amplitude modulated electromagnetic waves with the same frequency.
Expert Opin Investig Drugs. 2011 Aug;20(8):1099-106. doi: 10.1517/13543784.2011.583236. Epub 2011 May 9.
Tumor treating fields: concept, evidence and future.
Medical Oncology, Department of Internal Medicine, and Tumor Center, Kantonsspital Winterthur, Brauerstrasse, Switzerland. firstname.lastname@example.org
INTRODUCTION: Local control is fundamental, both for the curative as well as the palliative treatment of cancer. Tumor treating fields (TTFields) are low intensity (1 2 V/cm), intermediate frequency (100 ? 200 kHz) alternating electric fields administered using insulated electrodes placed on the skin surrounding the region of a malignant tumor. TTFields were shown to destroy cells within the process of mitosis via apoptosis, thereby inhibiting tumor growth. TTFields have no effect on non-dividing cells.
AREAS COVERED: This article reviews in vitro and in vivo preclinical studies, demonstrating the activity of TTFields both as a monotherapy as well as in combination with several cytotoxic agents. Furthermore, it summarizes the clinical experience with TTFields, mainly in two indications: one in recurrent glioblastoma multiforme: in a large prospective randomized Phase III trial TTFields was compared with best standard care (including chemotherapy): TTFields significantly improved median overall survival (OS) compared with standard therapy (7.8 vs 6.1 months) for the patients treated per protocol. Importantly, quality of life was also better in the TTFields group. The second indication was a Phase II study in second-line non-small cell lung cancer, where TTFields was administered concomitantly with pemetrexed. This combination resulted in an excellent median OS of 13.8 months. Interestingly, the progression-free survival (PFS) within the area of the TTFields was 28, however, outside the TTFields the PFS was only 22 weeks.
EXPERT OPINION: The proof of concept of TTFields has been well demonstrated in the preclinical setting, and the clinical data seem promising in various tumor types. The side effects of TTFields were minimal and in general consisted of skin reaction to the electrodes. There are a number of ways in which TTFields could be further evaluated, for example, in combination with chemotherapy, as a maintenance treatment, or as a salvage therapy if radiotherapy or surgery is not possible. While more clinical data are clearly needed, TTFields is an emerging and promising novel treatment concept.
Treatment of advanced hepatocellular carcinoma with very low levels of amplitude-modulated electromagnetic fields
Therapeutic options for patients with advanced hepatocellular carcinoma (HCC) are limited. There is emerging evidence that the growth of cancer cells may be altered by very low levels of electromagnetic fields modulated at specific frequencies.
A single-group, open-label, phase I/II study was performed to assess the safety and effectiveness of the intrabuccal administration of very low levels of electromagnetic fields amplitude modulated at HCC-specific frequencies in 41 patients with advanced HCC and limited therapeutic options. Three-daily 60-min outpatient treatments were administered until disease progression or death. Imaging studies were performed every 8 weeks. The primary efficacy end point was progression-free survival 6 months. Secondary efficacy end points were progression-free survival and overall survival.
Treatment was well tolerated and there were no NCI grade 2, 3 or 4 toxicities. In all, 14 patients (34.1%) had stable disease for more than 6 months. Median progression-free survival was 4.4 months (95% CI 2.1–5.3) and median overall survival was 6.7 months (95% CI 3.0–10.2). There were three partial and one near complete responses.
Treatment with intrabuccally administered amplitude-modulated electromagnetic fields is safe, well tolerated, and shows evidence of antitumour effects in patients with advanced HCC.
Treatment of inoperable or metastatic solid tumours is a major challenge in oncology, which is limited by the small number of therapeutic agents that are both well tolerated and capable of long-term control of tumour growth. Hepatocellular carcinoma (HCC) is the second most common cause of cancer death in men and the sixth in women worldwide (Jemal et al, 2011). Hepatocellular carcinoma is the most common tumour in certain parts of the world, particularly in East Asia, Africa, and certain countries of South America. This tumour is less frequent in Europe and in the United States, but has become the fastest rising cancer in the United States (Jemal et al, 2011). In the United States alone, it is estimated that 24120 new cases were diagnosed and there were 17430 deaths from HCC in 2010 (Jemal et al, 2010), a 27% increase in the number of new cases since 2004 (Jemal et al, 2004). The prognosis of patients suffering from advanced HCC is poor with an average survival of fewer than 6 months (Kassianides and Kew, 1987; Jemal et al, 2011).
Therapies for HCC are limited. Resections of the primary tumour or liver transplantation are the preferred therapeutic approaches in patients who are surgical candidates (Bruix and Sherman, 2005). Although these interventions result in long-term survival for some patients, only a minority benefit from them because of limitations due to tumour size, patient’s overall condition, and presence of hepatic cirrhosis (Cance et al, 2000). Only a small number of randomised trials show a survival benefit in the treatment of HCC. Chemoembolisation has been shown to confer a survival benefit in selected patients with unresectable HCC (Llovet et al, 2002). Data from two phase III randomised placebo-controlled studies demonstrate improved survival in patients with advanced HCC receiving the multikinase inhibitor sorafenib (Llovet et al, 2008b; Cheng et al, 2009). Additional therapies for this disease are sorely needed, especially for the large number of patients with advanced disease who cannot tolerate chemotherapy or intrahepatic interventions because of impaired liver function (Thomas and Zhu, 2005).
The intrabuccal administration of low and safe levels of electromagnetic fields, which are amplitude-modulated at disease-specific frequencies (RF AM EMF) (Figure 1), was originally developed for the treatment of insomnia (Pasche et al, 1990). The highest levels of EMFs encountered during treatment are found at the interface between the tongue and the mouth probe and are compliant with international safety limits (ICNIRP, 1998; Pasche and Barbault, 2003). Tumour-specific modulation frequencies have been identified for several common forms of cancer and one report suggests that this novel therapeutic approach is well tolerated and may be effective in patients with a diagnosis of cancer (Barbault et al, 2009). However, the safety and potential efficacy of this treatment approach in the treatment of advanced HCC are unknown. We designed this single-group, open-label, phase I/II study to assess the feasibility of this treatment in patients with advanced HCC and limited therapeutic options.
Patients and methods
The study was aimed at offering treatment to patients with Child–Pugh A or B advanced HCC and limited therapeutic options. Patients were classified as having advanced disease if they were not eligible for surgical resection or had disease progression after surgical or locoregional therapies or had disease progression after chemotherapy or sorafenib therapy. Patients with measurable, inoperable HCC were eligible for enrolment. Previous local or systemic treatments were allowed as long as they were discontinued at least 4 weeks before enrolment. Inclusion criteria included Eastern Cooperative Oncology Group performance status of 0, 1, or 2 and biopsy-confirmed HCC. Also allowed were patients with no pathological confirmation of HCC with a level of ?-fetoprotein higher than 400ngml?1 and characteristic imaging findings as assessed by multislice computer tomography (CT) scan or intravenous contrast ultrasound (US). As per the University of São Paulo Department of Transplantation and Liver Surgery guidelines, liver biopsies are avoided in patients eligible for transplant or with severely impaired liver function. Exclusion criteria included confirmed or suspected brain metastasis, Child–Pugh C, previous liver transplant, and pregnancy.
This was an investigator-initiated, single centre, uncontrolled phase I/II trial in patients with advanced HCC. The trial was approved by the local human investigation committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient. The protocol was registered: clinicaltrial.gov identifier no. NCT00534664.
Administration of AM EMFs
The generator of AM EMFs consists of a battery-driven radiofrequency (RF) EMF generator connected to a 1.5m long 50? coaxial cable, to the other end of which a stainless-steel spoon-shaped mouthpiece is connected via an impedance transformer (Figure 1A). The RF source of the device corresponds to a class C amplifier operating at 27.12MHz. The carrier frequency is AM (Figure 1B) with a modulation depth of 85±5%, whereas the modulation frequency is generated by a digital direct synthesiser with a resolution of 10?7. The treatment sequence is controlled by a microcontroller (Atmel AT89S8252, Fribourg, Switzerland), that is, duration of session, sequence of modulation frequencies and duration of each sequence can be programmed via PC over a RS232 interface. The RF output is adjusted to 100mW into a 50? load, which results in an emitting power identical to that of the device used for the treatment of insomnia (Pasche et al, 1990; Reite et al, 1994; Pasche et al, 1996). The United States Food and Drug Administration has determined that such a device is not a significant risk device and it has been used in several studies conducted in the United States (Reite et al, 1994; Pasche et al, 1996; Kelly et al, 1997). A long-term follow-up survey of 807 patients who have received this therapy in the United States, Europe and Asia showed that the rate of adverse reactions was low and was not associated with increases in the incidence of malignancy or coronary heart disease (Amato and Pasche, 1993). The maximum specific absorption rate (SAR) of the applied RF averaged over any 10g of tissue has been estimated to be less than 2Wkg?1, and the maximum temperature increase is significantly lower than 1°C anywhere in the body owing to RF absorption. The induced RF field values within the primary and metastatic tumours are significantly lower. In contrast, the RF fields induced during RF ablation of tumours cause hyperthermia and result in SAR in the range of 2.4 × 105Wkg?1 (Chang, 2003), that is, more than 100000 times higher than those delivered by the device used in this study.
We have previously reported the discovery of HCC-specific modulation frequencies in 46 patients with HCC using a patient-based biofeedback approach and shown the feasibility of using AM EMFs for the treatment of patients with cancer (Barbault et al, 2009). The treatment programme used in this study consisted of three-daily outpatient treatments of 1h duration, which contained HCC-specific modulation frequencies ranging between 100Hz and 21kHz administered sequentially, each for 3s (Figure 1C and Supplementary Table S1).
The treatment method consists of the administration of AM EMFs by means of an electrically conducting mouthpiece, which is in direct contact with the oral mucosa (Figure 1D). The patients were instructed on the use of the device and received the first treatment at the medical centre’s outpatient clinic. A device was provided to each patient for the duration of the study. The patients were advised to self-administer treatment three times a day. Treatment was administered until tumour progression was objectively documented. At that time, treatment was discontinued. Treatment compliance was assessed at every return visit by recording the number of treatments delivered in the preceding 2 months.
Efficacy end points and disease assessment
The primary end point of this trial was the proportion of patients progression-free at 6 months. Secondary end points were progression-free survival (PFS) (first day of treatment until progression of disease or death) and overall survival (OS) (first day of receiving treatment to death). Objective response was assessed using the Response Evaluation Criteria in Solid Tumours group classification for patients with disease assessed by either helical multiphasic CT (Therasse et al, 2000). Whenever contrast-enhanced US radiological assessment was used, it was performed and reviewed by the same radiologist specialised in HCC (MCC) as this imaging modality is investigator dependent. Tumour measurements were performed at baseline and every 8 weeks. Only patients with at least one repeat tumour measurement during therapy were considered for response analysis. Throughout the study, lesions measured at baseline were evaluated using the same technique (CT or contrast-enhanced US). Overall tumour response was scored as a complete response (CR), partial response (PR), or stable disease (SD) if the response was confirmed at least 4 weeks later. Alpha-fetoprotein (AFP) levels were measured every 8 weeks in all patients throughout the study, but changes in AFP were not an end point for assessment of response. Pain was assessed according to the NCI-CTCAE v.3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf).
Statistical analyses and efficacy assessment
All eligible patients who began treatment were considered assessable for the primary and secondary end points. A Simon two-stage phase II minimax design was used (Simon, 1989) to evaluate the rate of progression-free survival at 6 months. The interim analysis was performed once enrolment into the first stage was completed. In the first stage, 23 patients were observed. If two or fewer patients had progression-free survival 6 months, the trial would be terminated early for lack of efficacy. If the progression-free survival of 3 or more of the first 23 patients was equal or greater than 6 months, then an additional 18 patients would be enrolled to a maximum of 41 patients. If eight or more of the 41 had PFS of at least 6 months, we would conclude that the treatment was efficacious. This design had a Type I error rate of 5% and a Type II error rate of 10% for the null hypothesis of a 6-month PFS rate of 10% vs the alternative of 27.5%. Kaplan–Meier estimates of survival, PFS, and duration of response were calculated with standard errors based on Greenwood’s formula. These calculations were performed using the Proc Lifetest in SAS 9.2 (SAS Institute Inc., Cary, NC, USA).
Patient recruitment and follow-up
From October 2005 to July 2007, 267 patients were assessed for eligibility (Figure 2). In all, 43 patients with advanced HCC and Child–Pugh A or B were enrolled in this study. The date of last patient follow-up is 9 June 2011. Of these, 20 patients (46.5%) had histological confirmation of HCC; 23 patients (53.5%) were diagnosed based on elevated levels of ?-fetoprotein and characteristic imaging findings such as vascular invasion and characteristic differences in tumour blood flow. One patient was excluded because liver biopsy established the diagnosis of metastatic breast cancer. Another patient was excluded because of severely impaired liver function (Child–Pugh C11). These two patients who did not meet the inclusion criteria were registered as screening failures. Hence, a total of 41 patients were eligible to receive experimental therapy (Figure 2).
Two patients were lost to follow-up as they did not come back for their scheduled appointments. Repeated efforts were made to reach the patients and their families. The date of death of only one patient is known, and no information on response to treatment is available for either patient. Four patients withdrew consent while receiving therapy after 8.0, 9.3, 20.3, and 21.0 months, respectively (Figure 2). One patient elected to receive chemotherapy, one patient had poor treatment compliance as defined by administration of less than 50% of planned treatments at two consecutive return visits, one patient elected to enrol in another experimental protocol, and one patient requested to be considered for liver transplantation as part of an extended indication, which does not fulfil the Milan criteria (Mazzaferro et al, 1996). This latter patient experienced disease progression and was ultimately not eligible for liver transplantation. Of the 35 patients who discontinued experimental therapy, four died of gastrointestinal bleeding, three of sepsis, three of hepatic failure, one of chronic obstructive pulmonary disease, two of chemotherapy- and chemoembolisation-related complications, and one of myocardial infarction (Figure 2). The remaining 24 patients discontinued because of disease progression assessed by imaging or significant clinical deterioration as assessed by the investigator (Figure 2). Estimated 60-day mortality was 27.8% seven of 10 deaths were directly related to progression of disease. They were caused by liver failure in association with significant hepatic tumour involvement, without other cause of death, other than tumour involvement. Two deaths were secondary to gastrointestinal bleeding. One death was due to liver failure.
A total of 31 patients (75.6%) had radiological evidence of disease progression at the time of enrolment as defined by comparison of baseline imaging studies, with imaging studies obtained within the previous 6 months; 34 (82.9%) patients had received therapy before enrolment, five (14.6%) of them systemic chemotherapy or sorafenib (Table 1). Seven (17.1%) patients had not received therapy before enrolment for the following reasons: (1) severely impaired liver function in five cases; and (2) two patients refused to receive chemotherapy for metastatic disease. As shown in Table 2, the majority of patients had severely impaired liver function as demonstrated by the fact that 22 (53.7%) patients had Child–Pugh B disease and 35 (85.4%) BLCL stage C disease.
Six of the first 23 patients (26.1%) had progression-free survival 6 months, which led us to continue enrolling patients up to the preplanned total of 41 patients (Figure 2). In total, 14 patients (34.1%) had SD for more than 6 months, which met our preplanned primary efficacy end point. Median progression-free survival was 4.4 months (95% CI 2.1–5.3) and median OS was 6.7 months (95% CI 3.0–10.2) (Figure 3A and B). One patient, previously enrolled in the SHARP study (Llovet et al, 2008b) and with evidence of disease progression at the time of enrolment, remains on therapy with a near complete response for 58 months (Figure 3C). Estimated survival at 12, 24 and 36 months is 27.9% (s.e.=7.1%), 15.2% (s.e.=5.7%), and 10.1% (s.e.=4.8%), respectively. Subset analyses by Child-Pugh stage and accompanying figures are reported in Supplementary Information.
A total of 28 patients were evaluable for tumour response (Figure 2). Four (9.8%) patients had a partial response assessed with CT with or without contrast-enhanced ultrasound (Table 3). All partial responses were independently reviewed by two authors (MSR and DM). Three patients had biopsy-confirmed HCC and three had radiological evidence of disease progression at the time of enrolment (Table 4). Two patients had Child–Pugh A, one Child–Pugh B disease, and one had no cirrhosis. One of these patients without biopsy-proven disease subsequently withdrew consent after 4.9 months to undergo liver transplantation. The patient died of progression of disease 9.4 months later before undergoing liver transplantation. One patient with Child–Pugh B disease had a partial response lasting 11.7 months and died of gastrointestinal bleeding. One patient died of disease progression at 44.6 months. Overall, there were six long-term survivors with an OS greater than 24 months and four long-term survivors with an OS greater than 3 years. Importantly, five of the six (83%) long-term survivors had radiological evidence of disease progression at the time of study enrolment (Table 4). Two of three patients with the longest survival (44.6 and +58 months) had radiological evidence of disease progression at the time of enrolment, BLCL stage C disease, as well as portal vein thrombosis, three predictors of short survival (Llovet et al, 2003). Serial AFP measurements, which predict radiological response and survival in patients with HCC (Chan et al, 2009; Riaz et al, 2009), were available for 23 patients. AFP decreased by 20% or more in four (9.8%) patients following initiation of therapy (Table 5). Figure 3D shows the time course of a 37-fold decrease in AFP in a patient who had a long-lasting (11.7 months) partial response as assessed by CT.
In all, 11 patients reported pain before treatment initiation, 3 patients reported grade 3, 5 patients reported grade 2, and 3 patients grade 1. Five patients reported complete disappearance of pain and two patients reported decreased pain shortly after treatment initiation. Two patients reported no changes and two patients reported increased pain. There were no treatment-related grade 2, 3, or 4 toxicities. The only treatment-related adverse events were grade 1 mucositis (one patient) and grade 1 somnolence (one patient) over a total of 266.8 treatment months.
Treatment with AM EMFs did not show any significant toxicity despite long-term treatment. The lack of toxicity experienced by the 41 patients presented in this report as well as the 28 patients from our previous report (Barbault et al, 2009) can be readily explained by the very low and safe levels of induced RF EMFs, which are more than 100000 times lower than those delivered during RF ablation procedures (Chang, 2003). Hence, the putative mechanism of action of this novel therapeutic approach does not depend on temperature changes within the tumour.
These data are comparable to recent phase II studies evaluating the effectiveness of standard chemotherapy as well as novel targeted therapies in HCC (Abou-Alfa et al, 2006; Boige et al, 2007; Chuah et al, 2007; Cohn et al, 2008; Dollinger et al, 2008; Siegel et al, 2008). In a large phase II study assessing the effects of sorafenib in patients with HCC and Child–Pugh A and B who had not received previous systemic treatment, Abou-Alfa et al (2006) observed partial responses using the WHO criteria in 2.2% of patients. Investigator-assessed median time to progression was 4.2 months, and median OS was 9.2 months. Of note, all 137 patients from that study had evidence of disease progression after 14.8 months (Abou-Alfa et al, 2006), whereas, at the same time point, four (9.8%) of the patients enrolled in this study did not have evidence of disease progression. These findings suggest that RF AM EMF may increase the time to radiological progression in advanced HCC.
The majority of patients enrolled in this study had either failed standard treatment options or had severely impaired liver function that limited their ability to tolerate any form of systemic or intrahepatic therapy. Indeed, 16 patients (39.0%) had Child–Pugh B8 or B9 disease. Among these patients, the median progression-free survival was 4.4 months (95% CI 1.6–7.6 months), which is identical to that of the entire group. Five of these 16 patients (31.3%) received therapy for more than 7.5 months, which indicates that this therapy is well tolerated even in patients with severely impaired liver function.
Previous treatment with standard chemotherapy or sorafenib does not seem to impact the effectiveness of AM EMFs in the treatment of HCC. Indeed, three of the four patients who had a partial response while receiving AM EMFs had received previous systemic therapies (chemotherapy and sorafenib) and one had received intrahepatic therapy with 131I-lipiodol.
Tumour shrinkage as assessed by radiological imaging as well as changes in AFP levels were documented in patients with advanced HCC receiving RF EMF modulated at HCC-specific frequencies administered by an intrabuccal probe. Antitumour activity in patients with advanced HCC was exemplified by partial responses observed in four patients (9.8%) and decreases in AFP levels greater than 20% in four patients. A total of 18 patients (43.9%) either had objective response or SD 6 months.
Importantly, this therapeutic approach has long-lasting therapeutic effects in several patients with metastatic cancer. Two of these patients, one with recurrent thyroid cancer metastatic to the lungs (Figure 4) enrolled in our feasibility study (Barbault et al, 2009) and the patient shown in Figure 3C, are still receiving treatment without any evidence of disease progression and without side effects almost 5 years after being enrolled in these studies. These findings suggest that, in some patients, this therapeutic approach may achieve permanent control of advanced cancer with virtually no toxicity.
Our phase I/II study has several limitations. First, only 19 of the 41 patients had biopsy-proven HCC, and the others were diagnosed by clinical criteria, an approach similar to that used in a recently reported phase II trial evaluating the clinical and biological effects of bevacizumab in unresectable HCC (Siegel et al, 2008). Importantly, analysis restricted to these 19 patients shows rates of progression-free survival at 6 months, median progression-free survival and OS that are similar to those without biopsy-proven HCC (Supplementary Figures 1C and D). Furthermore, three of the four partial responses were observed in patients with biopsy-proven HCC. Hence, these findings strongly suggest that treatment with AM EMFs yields similar results in patients with and without biopsy-confirmed HCC. Another potential limitation of our study consists in the use of contrast-enhanced ultrasound for the monitoring of some patients with HCC. It should be pointed out that recent studies indicate that the use of this imaging technique is comparable to that of CT scan with respect to the measurement of HCC tumours (Choi, 2007; Maruyama et al, 2008).
Antitumour response is considered the primary end point for phase II studies to proceed to further investigations. Studies applying Cox proportional hazards analysis indicate that this end point is consistently associated with survival in trials of locoregional therapies for HCC (Llovet et al, 2002) and a recent consensus article suggests that randomised studies are necessary to capture the true efficacy of novel therapies in HCC (Llovet et al, 2008a). In summary, the encouraging findings from this study warrant a randomised study to determine the impact of AM EMFs on OS and time to symptomatic progression.
We thank Drs Al B Benson III, Northwestern University and Leonard B Saltz, Memorial Sloan-Kettering Cancer Center for reviewing the manuscript.
AB and BP have filed a patent related to the use of electromagnetic fields for the diagnosis and treatment of cancer. AB and BP are founding members of TheraBionic LLC.
Supplementary Information accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)
Supplementary Figure 1
Supplementary Figure 2
Supplementary Table 1
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Electromagn Biol Med. 2010 Dec;29(4):132-43.
Bioelectromagnetic field effects on cancer cells and mice tumors.
Laboratory Bioelectrochemistry, Beutenberg Campus, Jena, Germany.
We present possibilities and trends of ELF bioelectromagnetic effects in the mT amplitude range on cancer cells and on mice bearing tumors. In contrast to invasive electrochemotherapy and electrogenetherapy, using mostly needle electrodes and single high-amplitude electropulses for treatment, extremely low-frequency (ELF) pulsating electromagnetic fields (PEMF) and sinusoidal electromagnetic fields (SEMF) induce tumor cell apoptosis, inhibit angiogenesis, impede proliferation of neoplastic cells, and cause necrosis non invasively, whereas human lymphocytes are negligibly affected. Our successful results in killing cancer cells-analyzed by trypan blue staining or by flow cytometry-and of the inhibition of MX-1 tumors in mice by 15-20?mT, 50?Hz treatment in a solenoid coil also in the presence of bleomycin are presented in comparison to similar experimental results from the literature. In conclusion, the synergistic combinations of PEMF or SEMF with hyperthermia (41.5°C) and/or cancerostatic agents presented in the tables for cells and mice offer a basis for further development of an adjuvant treatment for patients suffering from malignant tumors and metastases pending the near-term development of suitable solenoids of 45-60?cm in diameter, producing >20?mT in their cores.
Focusing properties of picosecond electric pulses in non-invasive cancer treatment.
State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China. email@example.com
In the light of optical theory, we advanc an ultra-wideband impulse radiating antenna (IRA) which is composed of an ellipsoidal reflector and a cone radiator. The high-intensity ultra-short electric pulses radiated by IRA can be transferred into the deep target in tissue non-invasively and be focused effectively. With the focused picosecond electric pulses, the organelles (mitochondria) transmembrane potential shall change to collapse under which the tumor cells will be targetly induced to apoptosis, so the method of non-invasive treatment of tumors would be achieved. Based on the time-domain electromagnetic field theory, the propagation characteristics of picosecond electric pulses were analyzed with and without the context of biological tissue, respectively. The results show that the impulse characteristics of input pulse were maintained and the picosecond electric pulses can keep high resolution in target areas. Meanwhile, because of the dispersive nature of medium, the pulse amplitude of the pulses will attenuate and the pulse width will be broadened.
BMC Cancer. 2010 Apr 24;10:159.
Anti-proliferative effect of extremely low frequency electromagnetic field on preneoplastic lesions formation in the rat liver.
Jiménez-García MN, Arellanes-Robledo J, Aparicio-Bautista DI, Rodríguez-Segura MA, Villa-Treviño S, Godina-Nava JJ.
Department of Physics Center of Research and Advanced Studies of the National Polytechnic Institute, Mexico City, Mexico. firstname.lastname@example.org
BACKGROUND: Recently, extremely low frequency electromagnetic fields (ELF-EMF) have been studied with great interest due to their possible effects on human health. In this study, we evaluated the effect of 4.5 mT-120 Hz ELF-EMF on the development of preneoplastic lesions in experimental hepatocarcinogenesis.
METHODS: Male Fischer-344 rats were subjected to the modified resistant hepatocyte model and were exposed to 4.5 mT – 120 Hz ELF-EMF. The effects of the ELF-EMF on hepatocarcinogenesis, apoptosis, proliferation and cell cycle progression were evaluated by histochemical, TUNEL assay, caspase 3 levels, immunohistochemical and western blot analyses.
RESULTS: The application of the ELF-EMF resulted in a decrease of more than 50% of the number and the area of gamma-glutamyl transpeptidase-positive preneoplastic lesions (P = 0.01 and P = 0.03, respectively) and glutathione S-transferase placental expression (P = 0.01). The number of TUNEL-positive cells and the cleaved caspase 3 levels were unaffected; however, the proliferating cell nuclear antigen, Ki-67, and cyclin D1 expression decreased significantly (P < or = 0.03), as compared to the sham-exposure group.
CONCLUSION: The application of 4.5 mT-120 Hz ELF-EMF inhibits preneoplastic lesions chemically induced in the rat liver through the reduction of cell proliferation, without altering the apoptosis process.
Cell Biochem Biophys. 2009;55(1):25-32. Epub 2009 Jun 18.
Evaluation of the potential in vitro antiproliferative effects of millimeter waves at some therapeutic frequencies on RPMI 7932 human skin malignant melanoma cells.
Department of Chemistry, University of Calabria, Via P. Bucci, Cubo 17/D, Arcavacata di Rende (CS), Italy.email@example.com
The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm(2) were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were < or = 0.3 mW/cm(2). Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.
Bioelectrochemistry. 2010 Oct;79(2):257-60. Epub 2010 Mar 10.
Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells.
Traitcheva N, Berg H.
Institute of Plant Physiology “M. Popov,” Bulgarian Acad. of Sciences, Sofia, Bulgaria.
In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determined by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths lambda > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicin). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo=1.4 kV/cm and pulse time constants in the time range 0.5-3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U<100 V, I=55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.
Arch Biochem Biophys. 2010 May;497(1-2):82-9. Epub 2010 Mar 24.
Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma.
Ford WE, Ren W, Blackmore PF, Schoenbach KH, Beebe SJ.
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.
Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.
J Physiol Pharmacol. 2010 Apr;61(2):201-5.
Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.
Kaszuba-Zwoinska J, Wojcik K, Bereta M, Ziomber A, Pierzchalski P, Rokita E, Marcinkiewicz J, Zaraska W, Thor P.
Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland. firstname.lastname@example.org
Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.
Int J Radiat Biol. 2010 Feb;86(2):79-88.
Growth of injected melanoma cells is suppressed by whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields.
Hu JH, St-Pierre LS, Buckner CA, Lafrenie RM, Persinger MA.
Department of Biology, Laurentian University, Sudbury, Ontario, Canada.
PURPOSE: To measure the effect of exposure to a specific spatial-temporal, hysiologically-patterned electromagnetic field presented using different geometric configurations on the growth of experimental tumours in mice.
METHODS: C57b male mice were inoculated subcutaneously with B16-BL6 melanoma cells in two blocks of experiments separated by six months (to control for the effects of geomagnetic field). The mice were exposed to the same time-varying electromagnetic field nightly for 3 h in one of six spatial configurations or two control conditions and tumour growth assessed.
RESULTS: Mice exposed to the field that was rotated through the three spatial dimensions and through all three planes every 2 sec did not grow tumours after 38 days. However, the mice in the sham-field and reference controls showed massive tumours after 38 days. Tumour growth was also affected by the intensity of the field, with mice exposed to a weak intensity field (1-5 nT) forming smaller tumours than mice exposed to sham or stronger, high intensity (2-5 microT) fields. Immunochemistry of tumours from those mice exposed to the different intensity fields suggested that alterations in leukocyte infiltration or vascularisation could contribute to the differences in tumour growth.
CONCLUSIONS: Exposure to specific spatial-temporal regulated electromagnetic field configurations had potent effects on the growth of experimental tumours in mice.
Melanoma Res. 2009 Aug 26. [Epub ahead of print]
Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.
Chen X, James Swanson R, Kolb JF, Nuccitelli R, Schoenbach KH.
Department of Hepatobiliary Surgery, the First Affiliated Hospital Zhejiang University School of Medicine, Hangzhou, Zhejiang, China bFrank Reidy Research Center for Bioelectrics cDepartment of Biological Sciences, Old Dominion University, Norfolk, Virginia, USA.
Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.
Cancer Biol Ther. 2009 Sep;8(18):1756-62. Epub 2009 Sep 17.
Static magnetic fields impair angiogenesis and growth of solid tumors in vivo.
Strelczyk D, Eichhorn ME, Luedemann S, Brix G, Dellian M, Berghaus A, Strieth S.
Walter-Brendel-Center for Experimental Medicine (WBex), Campus Grosshadern, University of Munich (LMU), Munich, Germany.
Exposure to static magnetic fields (SMFs) results in a reduced blood flow in tumor vessels as well as in activation and adherence of platelets. Whether this phenomenon may have a significant functional impact on tumors has not been investigated as yet. The aim of our study was to evaluate the effects of prolonged exposure to SMFs on tumor angiogenesis and growth. Experiments were performed in dorsal skinfold chamber preparations of Syrian Golden hamsters bearing syngenic A-Mel-3 melanomas. On 3 d following tumor cell implantation one group of animals was immobilized and exposed to a SMF of 586 mT for three h. Control animals were immobilized for the same duration without SMF exposure. Using in vivo-fluorescence microscopy the field effects on tumor angiogenesis and microcirculation were analyzed for seven days. Tumor growth was assessed by repeated planimetry of the tumor area during the observation period. Exposure to SMFs resulted in a significant retardation of tumor growth ( approximately 30%). Furthermore, histological analysis showed an increased peri- and intratumoral edema in tumors exposed to SMFs. Analysis of microcirculatory parameters revealed a significant reduction of functional vessel density, vessel diameters and red blood cell velocity in tumors after exposure to SMFs compared to control tumors. These changes reflect retarded vessel maturation by antiangiogenesis. The increased edema after SMF exposure indicates an increased tumor microvessel leakiness possibly enhancing drug-uptake. Hence, SMF therapy appears as a promising new anticancer strategy-as an inhibitor of tumor growth and angiogenesis and as a potential sensitizer to
J Exp Clin Cancer Res. 2009 Apr 14;28:51.
Amplitude-modulated electromagnetic fields for the treatment of cancer: discovery of tumor-specific frequencies and assessment of a novel therapeutic approach.
Barbault A, Costa FP, Bottger B, Munden RF, Bomholt F, Kuster N, Pasche B.
Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus.
Hu YW, Liu CY, Du CM, Zhang J, Wu WQ, Gu ZL.
Department of Pharmacology, Medical College of Soochow University, 199 RenAi Road, Suzhou 215123, PR China.
AIM OF THE STUDY: Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.
MATERIALS AND METHODS: Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.
RESULTS: The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.
CONCLUSIONS: These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.
Amplitude-modulated electromagnetic fields for the treatment of cancer: Discovery of tumor-specific frequencies and assessment of a novel therapeutic approach
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Because in vitro studies suggest that low levels of electromagnetic fields may modify cancer cell growth, we hypothesized that systemic delivery of a combination of tumor-specific frequencies may have a therapeutic effect. We undertook this study to identify tumor-specific frequencies and test the feasibility of administering such frequencies to patients with advanced cancer.
Patients and methods
We examined patients with various types of cancer using a noninvasive biofeedback method to identify tumor-specific frequencies. We offered compassionate treatment to some patients with advanced cancer and limited therapeutic options.
We examined a total of 163 patients with a diagnosis of cancer and identified a total of 1524 frequencies ranging from 0.1 Hz to 114 kHz. Most frequencies (57–92%) were specific for a single tumor type. Compassionate treatment with tumor-specific frequencies was offered to 28 patients. Three patients experienced grade 1 fatigue during or immediately after treatment. There were no NCI grade 2, 3 or 4 toxicities. Thirteen patients were evaluable for response. One patient with hormone-refractory breast cancer metastatic to the adrenal gland and bones had a complete response lasting 11 months. One patient with hormone-refractory breast cancer metastatic to liver and bones had a partial response lasting 13.5 months. Four patients had stable disease lasting for +34.1 months (thyroid cancer metastatic to lung), 5.1 months (non-small cell lung cancer), 4.1 months (pancreatic cancer metastatic to liver) and 4.0 months (leiomyosarcoma metastatic to liver).
Cancer-related frequencies appear to be tumor-specific and treatment with tumor-specific frequencies is feasible, well tolerated and may have biological efficacy in patients with advanced cancer.
clinicaltrials.gov identifier NCT00805337
We have previously shown that the intrabuccal administration of low and safe levels of electromagnetic fields, amplitude-modulated at a frequency of 42.7 Hz by means of a battery-powered portable device modifies the electroencephalographic activity of healthy subjects [1,2], and is associated with subjective and objective relaxation effects . We have also shown that sequential administration of four insomnia-specific frequencies, including 42.7 Hz, results in a significant decrease in sleep latency and a significant increase in total sleep time in patients suffering from chronic insomnia [4,5]. This approach has been termed Low Energy Emission Therapy (LEET). Dosimetric studies have shown that the amount of electromagnetic fields delivered to the brain with this approach is 100 to 1000 times lower than the amount of electromagnetic fields delivered by handheld cellular phones and does not result in any heating effect within the brain . The U.S. FDA has determined that such a device is not a significant risk device. A long-term follow-up survey of 807 patients who have received this therapy in the U.S., Europe and Asia revealed that the rate of adverse reactions were low and were not associated with increases in the incidence of malignancy or coronary heart disease .
While many discoveries in medicine have evolved from a scientific rationale based on in vitro and in vivo findings, several seminal discoveries are the results of biological effects first observed in humans. For example, the development of modern cancer chemotherapy can be traced directly to the clinical observation that individuals exposed to mustard gas, a chemical warfare agent, had profound lymphoid and myeloid suppression. These observations led Goodman and Gilman to use this agent to treat cancer. Given the advantageous safety profile of athermal, non-ionizing radiofrequency electromagnetic fields and the emerging evidence that low levels of electromagnetic or electric fields may modify the growth of tumor cells [9–11], we hypothesized that the growth of human tumors might be sensitive to different but specific modulation frequencies. We tested this hypothesis through examination of a large number of patients with biopsy-proven cancer. Using a patient-based biofeedback approach we identified strikingly similar frequencies among patients with the same type of cancer and observed that patients with a different type of cancer had biofeedback responses to different frequencies. These findings provided strong support for our initial hypothesis. Following identification of tumor-specific frequencies in 163 patients with a diagnosis of cancer, we offered compassionate treatment to 28 patients with advanced cancer and limited palliative therapeutic options. We are reporting the results of our frequency discovery studies as well as the results of a feasibility study making use of Low Energy Emission Therapy in the treatment of cancer.
Frequency discovery consists in the measurement of variations in skin electrical resistance, pulse amplitude and blood pressure. These measurements are conducted while individuals are exposed to low and safe levels of amplitude-modulated frequencies emitted by handheld devices. Exposure to these frequencies results in minimal absorption by the human body, which is well below international electromagnetic safety limits [12,13]. Patients are lying on their back and are exposed to modulation frequencies generated by a frequency synthesizer as described below. Variations in the amplitude of the radial pulse were used as the primary method for frequency detection. They were defined as an increase in the amplitude of the pulse for one or more beats during scanning of frequencies from 0.1 to 114,000 Hz using increments of 100 Hz. Whenever a change in the amplitude of the pulse is observed, scanning is repeated using increasingly smaller steps, down to 10-3 Hz. Frequencies eliciting the best biofeedback responses, defined by the magnitude of increased amplitude and/or the number of beats with increased amplitude, were selected as tumor-specific frequencies.
During our initial search for frequencies in patients with a diagnosis of cancer, we identified frequencies in the 1,000 to 15,000 Hz range. The range of these frequencies was higher than the frequencies previously identified in patients with insomnia (< 300 Hz). To enable the administration of well defined signals at these higher frequencies, the signal synthesizer used in the insomnia studies was redesigned and its accuracy verified at the laboratories of the Foundation for Research on Information Technology in Society (IT’IS, Zurich, Switzerland). The Direct Digital Synthesis (DDS) based synthesizer AD9835 (Analog Devices, Norwood, MA) with a frequency precision of 10-7 was used for frequency detection in patients with a diagnosis of cancer. Subsequently, the same frequency synthesizer was used for treatment administration. The concept of this novel device is depicted in Figure ?Figure11.
Generation of amplitude-modulated electromagnetic fields: the device consists of a battery-driven radiofrequency (RF) electromagnetic field generator connected to a 1.5 meter long 50 Ohm coaxial cable, to the other end of which a spoon-shaped mouthpiece made of steel is connected with the inner conductor. The RF source of the device corresponds to a high-level amplitude-modulated class C amplifier operating at 27.12 MHz. The modulation frequency can be varied between 0.01 Hz and 150 kHz with a modulation depth of 85 ± 5%. The output signal is controlled by a microcontroller AT89S8252 (Atmel, Fribourg, Switzerland), i.e. duration of a session, sequence of modulation frequencies, and duration of each sequence are programmed prior to the treatment with a PC connected to the panel of the device. The RF output is adjusted to 100 mW into a 50 Ohm load using a sinusoidal modulated test signal, which results in an emitting power identical to that of the device used in the treatment of insomnia [4,5].
Following a period of search and discovery of novel tumor-specific frequencies, outpatient treatment of patients with advanced cancer was initiated in Switzerland and Brazil on a compassionate basis, free of charge. Patients self-administered treatment for 60 min, three times a day. Oral informed consent was provided by seven patients. All other patients signed a written informed consent approved by a local human subject committee in compliance with the Helsinki declaration and the protocol was registered, clinicaltrials.gov identifier # NCT00805337. All patients had histologically confirmed diagnosis of cancer. Except for patients with a diagnosis of ovarian cancer, measurable disease was required. For patients with ovarian cancer, CA 125 level was used as a surrogate marker of measurable disease and a 50% increase in baseline level considered evidence of disease progression. All anticancer therapies had to be discontinued for at least one month prior to treatment initiation. Other eligibility criteria included an Eastern Cooperative Group performance status (PS) of 0 to 2 and an estimated life expectancy of at least 3 months.
Objective response in patients with measurable disease was assessed using the Response Evaluation Criteria in Solid Tumors group classification . Two of us (B.B. and R.F.M.) independently reviewed all imaging studies.
Patients were evaluated for treatment-related toxicity at a minimum every two months as per the National Cancer Institute Common Toxicity Criteria version 2.0. The worst grade of toxicity per patient was recorded.
A total of 115 patients were examined in Switzerland, 48 in Brazil (Table ?(Table1).1). There were 76 females and 87 males. The median age was 59 years (range 19 – 84). The most common tumor types were hepatocellular carcinoma (46), breast cancer (32), colorectal cancer (19), and prostate cancer (17).
Compassionate treatment with tumor-specific frequencies was offered to 28 patients (Table ?(Table2).2). Twenty six patients were treated in Switzerland and two patients were treated in Brazil. All patients were white, and 63% (n = 17) were female. Patients ranged in age from 30 to 82 years (median, 61 years) and 75% (n = 21) had PS of 1 (vs 0 or 2). Seventy-nine percent (n = 22) of patients had received at least one prior systemic therapy, 57% (n = 17) had received at least two prior systemic therapies (Table ?(Table22).
Once disease progression was observed, most patients elected to resume or initiate chemotherapy and/or targeted therapy. Seven (25%) patients requested to continue experimental treatment in combination with chemotherapy. Continuation of experimental treatment was allowed if desired by the patient and approved by the patient’s oncologist.
Discovery of tumor-specific frequencies
The exact duration of each examination was not recorded but lasted on average three hours. Each patient was examined an average of 3.3 ± 3.4 times (range 1 – 26). Frequency discovery was performed in patients with disease progression, stable disease or partial response. In general, we found more frequencies in patients with evidence of disease progression and large tumor bulk than in patients with stable disease, small tumor bulk or evidence of response. When we restrict the analysis of patients examined in 2006 and 2007, i.e. at a time when we had gathered more than 80% of the common tumor frequencies, we found that patients with evidence of disease progression had positive biofeedback responses to 70% or more of the frequencies previously discovered in patients with the same disease. Conversely, patients with evidence of response to their current therapy had biofeedback responses to 20% or less of the frequencies previously discovered in patients with the same disease. We also observed that patients examined on repeated occasions developed biofeedback responses to an increasing number of tumor-specific frequencies over time whenever there was evidence of disease progression. Whenever feasible, all frequencies were individually retested at each frequency detection session. These findings suggest that a larger number of frequencies are identified at the time of disease progression.
A total of 1524 frequencies ranging from 0.1 to 114 kHz were identified during a total of 467 frequency detection sessions (Table ?(Table1).1). The number of frequencies identified in each tumor type ranges from two for thymoma to 278 for ovarian cancer. Overall, 1183 (77.6%) of these frequencies were tumor-specific, i.e. they were only identified in patients with the same tumor type. The proportion of tumor-specific frequencies ranged from 56.7% for neuroendocrine tumors to 91.7% for renal cell cancer. A total of 341 (22.4%) frequencies were common to at least two different tumor types. The number of frequencies identified was not proportional to either the total number of patients studied or the number of frequency detection sessions (Table ?(Table11).
Treatment with tumor-specific amplitude-modulated electromagnetic fields
Twenty eight patients received a total of 278.4 months of experimental treatment. Median treatment duration was 4.1 months per patient; range 1 to +50.5. Patients treated in Switzerland were re-examined on average every other month for frequency detection; patients treated in Brazil were only examined once. Novel frequencies discovered upon re-examination were added to the treatment program of patients receiving experimental treatment. The first treatment programs consisted of combinations of less than ten frequencies while the most recent treatment programs exceed 280 frequencies (Figure ?(Figure22).
The evolution of treatment programs through incremental addition of tumor-specific frequencies is illustrated by the case of a 51 year old woman with ovarian cancer. This patient was diagnosed with FIGO stage III (G2–G3) ovarian cancer in October 1997 and had received multiple courses of palliative chemotherapy until 2005. As seen on Figure ?Figure2,2, the initial treatment consisting of 15 frequencies did not yield any response. Upon re-examination, 11 additional frequencies (total of 26) were added to the treatment program in August 05. Because of disease progression, treatment with single agent bevacizumab was initiated in November 05. Interestingly, the CA 125 level had decreased by 200 units between October and November 2005, prior to the initiation of bevacizumab. Combined treatment with amplitude-modulated electromagnetic fields and bevacizumab resulted in a decrease in CA 125 level from 2140 to 540 in May 06. Treatment was supplemented with cyclophosphamide from March to September 07. The patient was hospitalized with pneumonia and elected to only receive amplitude-modulated electromagnetic fields since September 07. As of April 1, 2009 the patient has stable disease and is asymptomatic. She has been receiving experimental treatment without interruption for a total of +50.5 months.
This case provides empirical evidence that adding tumor-specific frequencies may yield disease stabilization in patients with evidence of disease progression. However, addition of frequencies over time does not appear to be a requirement for therapeutic efficacy. This is illustrated by the case of a 59 yo postmenopausal female with ER/PR positive, ERBB2 negative breast cancer with biopsy confirmed metastasis to the left ischium and right adrenal gland (Figure ?(Figure3A,3A, Figure ?Figure3C,3C, Figure ?Figure3D).3D). She had been previously treated with radiation therapy to the left ischium, had received five different hormonal manipulations (tamoxifen, anastrozole, exemestane, fulvestran and megestrol). She had also received capecitabine, which had been discontinued because of gastrointestinal side effects. The patient was examined only once. In June 2006, at the time of treatment initiation, the patient complained of severe left hip pain, which was limiting her mobility despite the intake of opioids. Within two weeks of experimental treatment initiation with breast cancer-specific frequencies, the patient reported complete disappearance of her pain and discontinued the use of pain medications. She also reported a significant improvement in her overall condition. As seen on Figure ?Figure3B3B and ?and3E,3E, PET-CT obtained three months after treatment initiation showed complete disappearance of the right adrenal and left ischium lesions. The complete response lasted 11 months. Intriguingly, the patient had developed intermittent vaginal spotting in the months preceding experimental treatment initiation. A minimally enhancing uterine lesion was observed on PET-CT prior to treatment initiation. Upon follow-up, FDG uptake increased significantly (Figure ?(Figure3B)3B) and the patient was diagnosed with uterine cancer by hysteroscopy. The patient underwent hysterectomy, which revealed endometrial adenocarcinoma. Hence, while treatment with breast cancer specific frequencies resulted in a complete response, it did not affect the growth of endometrial adenocarcinoma. This observation suggests that breast cancer frequencies are tumor-specific as a response of the metastatic breast cancer was observed while a uterine tumor progressed.
As seen in Table ?Table3,3, sixteen patients were evaluable for response by RECIST criteria. A complete response was observed in a patient with hormone-refractory breast cancer metastatic to the adrenal gland and bone (Figure ?(Figure3),3), which lasted 11 months. A partial response was observed in a patient with hormone-refractory breast cancer metastatic to bone and liver, which lasted 13.5 months. Five patients had stable disease for +34.1 months (thyroid cancer with biopsy-proven lung metastases), 6.0 months (mesothelioma metastatic to the abdomen), 5.1 months (non-small cell lung cancer), and 4.1 months (pancreatic cancer with biopsy-proven liver metastases). As of April 1, 2009 two patients are still receiving experimental treatment and four patients are alive.
Adverse and beneficial reactions
No patients receiving experimental therapy reported any side effect of significance and no patient discontinued treatment because of adverse effects. Three patients (10.7%) reported grade I fatigue after receiving treatment. One patient (3.6%) reported grade I mucositis after long-term use (26 months) of the experimental device and concomitant chemotherapy. Two patients with severe bony pain prior to initiation of experimental treatment reported significant symptomatic improvement. Both patients had breast cancer metastatic to the skeleton.
In this report we describe the discovery of tumor-specific amplitude modulation frequencies in patients with a diagnosis of cancer using noninvasive biodfeedback methods. Our approach represents a significant departure from the development of novel forms of chemotherapy and targeted therapy, which commonly rely on in vitro and animal experiments, followed by phase I studies to assess tolerability. Given the absence of theoretical health risks related to the administration of very low level of electromagnetic fields and the excellent safety profile observed in patients suffering from insomnia treated for up to several years , our approach was entirely patient-based. This allowed us to examine a large number of patients with tumor types commonly encountered in Switzerland and Brazil. It also allowed us to examine the same patients on multiple occasions, which decreased the variability inherent to a single frequency detection session.
Examination of patients with cancer led to the identification of frequencies that were either specific for a given tumor type or common to two or more tumor types. We observed that most frequencies were tumor-specific. Indeed, when the analysis of frequencies is restricted to tumor types analyzed following a minimum of 60 frequency detection sessions (breast cancer, hepatocellular carcinoma, ovarian cancer and prostate cancer), at least 75% of frequencies appear to be tumor-specific. Some frequencies such as 1873.477 Hz, 2221.323 Hz, 6350.333 Hz and 10456.383 Hz are common to the majority of patients with a diagnosis of breast cancer, hepatocellular carcinoma, prostate cancer and pancreatic cancer. The small number of frequency detection sessions conducted in patients with thymoma, leiomyosarcoma, and bladder cancer constitutes a limitation of our study and an accurate estimate of tumor-specific versus nonspecific frequencies cannot yet be provided for these tumor types. Only one patient with thyroid cancer metastatic to the lung was examined 14 times over the course of the past three years and this led to the discovery of 112 frequencies, 79.5% of which were thyroid cancer-specific. These combined findings strongly suggest that many tumor types have a proportion of tumor-specific frequencies of more than 55%. The high number of frequencies observed in patients with ovarian cancer may be due to the various histologies associated with this tumor type.
We observed excellent compliance with this novel treatment as patients were willing to self-administer experimental treatment several times a day. The only observed adverse effects in patients treated with tumor-specific frequencies were grade I fatigue after treatment (10.6%) and grade I mucositis (3.6%). Fatigue was short-lived and no patient reported persistent somnolence. Of note, mucositis only occurred concomitantly with the administration of chemotherapy. The frequency and severity of adverse effects is analogous to what was observed in patients treated with insomnia-specific frequencies  and confirm the feasibility of this therapeutic approach and its excellent tolerability.
We did not observe any untoward reaction in patient receiving either chemotherapy or targeted therapy in combination with amplitude-modulated electromagnetic fields. While these latter findings are limited to 7 patients, they are consistent with the lack of theoretical interaction between very low level of electromagnetic fields and anticancer therapy. Furthermore, one patient received palliative radiation therapy concomitantly with experimental therapy without any adverse effects. These findings provide preliminary data suggesting that amplitude-modulated electromagnetic fields may be added to existing anticancer therapeutic regimens.
The objective responses observed suggest that electromagnetic fields amplitude-modulated at tumor-specific frequencies may have a therapeutic effect. Of the seven patients with metastatic breast cancer, one had a complete response lasting 11 months, another one a partial response lasting 13.5 months. These data provide a strong rationale to further study this novel therapy in breast cancer. The increased knowledge of tumor-specific frequencies and the preliminary evidence that additional tumor-specific frequencies may yield a therapeutic benefit (Figure ?(Figure2)2) provides a strong rationale for the novel concept that administration of a large number of tumor-specific frequencies obtained through the follow-up of numerous patients may result in long-term disease control. This hypothesis is partially supported by two long-term survivors reported in this study, a patient with thyroid cancer metastatic to the lung with stable disease for +34.1 months and a heavily pretreated patient with ovarian carcinoma and peritoneal carcinomatosis with stable disease for +50.5 months. Additional support for this hypothesis stems from the observation that four patients with advanced hepatocellular carcinoma in a follow-up phase II study by Costa et al had a partial response, two of them lasting more than 35 months. These exciting results provide hope that this novel therapeutic approach may yield long-term disease control of advanced cancer.
Kirson et al have recently reported the use of continuous wave (CW) electric fields between 100 KHz to 1 MHz [10,11]. These fields were CW, applied at relative high field strengths but lower frequencies than the fields used in our study. These frequencies were found to be effective when applied by insulating external electrodes to animal cancer models and patients with recurrent glioblastoma. In contrast to our approach, the electric fields applied to cancer cells and patients did not include any amplitude modulation. Hence, it is likely that these two different therapeutic modalities have different mechanisms of action.
Computer simulation studies have shown that the specific absorption rate (SAR) in the head resulting from the use of intrabuccally-administered amplitude-modulated electromagnetic fields is in the range of 0.1–100 mW/kg. Hence, the SAR outside the head is substantially below 0.1 mW/kg. We had previously hypothesized that the mechanism of action of electromagnetic fields amplitude-modulated at insomnia-specific frequencies was due to modification in ions and neurotransmitters, as demonstrated in animal models, as such biological effects had been reported at comparable SARs. However, this hypothesis does not provide a satisfactory explanation for the clinical results observed in patients with advanced cancer. First, the levels of electromagnetic fields delivered to organs such as the liver, adrenal gland, prostate and hip bones, are substantially lower than the levels delivered to the head. Second, there is currently no acceptable rationale for a systemic anti-tumor effect that would involve subtle changes in neurotransmitters and ions within the central nervous system. Consequently, we hypothesize that the systemic changes (pulse amplitude, blood pressure, skin resistivity) observed while patients are exposed to tumor-specific frequencies are the reflection of a systemic effect generated by these frequencies. These observations suggest that electromagnetic fields, which are amplitude-modulated at tumor-specific frequencies, do not act solely on tumors but may have wide-ranging effects on tumor host interactions, e.g. immune modulation. The exciting results from this study provide a strong rationale to study the mechanism of action of tumor-specific frequencies in vitro and in animal models, which may lead to the discovery of novel pathways controlling cancer growth.
AB and BP have filed a patent related to the use of electromagnetic fields for the diagnosis and treatment of cancer. AB and BP are founding members of TheraBionic LLC.
BP and AB conceived and designed the study. FB and NK designed the device and performed the EM dosimetry. AB, BP and FC collected and assembled the data. BB and RF independently reviewed the imaging studies. AB, BP and FC analyzed and interpreted the data. BP wrote the manuscript. All co-authors read and approved the final manuscript.
The authors would like to thank Al B. Benson, III, Northwestern University and Leonard B. Saltz, Memorial Sloan-Kettering Cancer Center for providing insightful comments and reviewing the manuscript. Neither of them received any compensation for their work. Presented in part: abstract (ID 14072) ASCO 2007; oral presentation (29th Annual Meeting of the Bioelectromagnetics Society, Kanazawa, Japan, 2007). Funding: study funded by AB and BP. The costs associated with the design and engineering of the devices used in this study were paid by AB and BP. BB and RM did not receive any compensation for their independent review of the imaging studies.
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Anticancer Res. 2008 Jul-Aug;28(4B):2245-51.
Effect of steep pulsed electric field on proliferation, viscoelasticity and adhesion of human hepatoma SMMC-7721 cells.
Song G, Qin J, Yao C, Ju Y.
Department of Bioengineering, College of Bioengineering, Ministry of Education of China, Chongqing University, Chongqing, PR China.
It has been proven that steep pulsed electric field (SPEF) can directly kill tumor cells and plays an important role in anticancer treatment. The biorheological mechanisms, however, that destroy tumor cells are almost unknown. To resolve this issue, here, an SPEF generator was used to assess the effects of high- and low-dose SPEF on the proliferation of human hepatoma SMMC-7721 cells by MTT assay, and on the viscoelasticity, adhesion of SMMC-7721 cells to endothelial cells by micropipette aspiration technique. Viability and proliferation of SPEF-treated SMMC-7721 cells were significantly inhibited. Cell cycle analysis indicated that SPEF arrested the cell cycle progression of SMMC-7721 cells at the G0/G1 transition to the S-phase. Viscoelastic data fitted by a standard linear solid model showed that viscoelasticity of SMMC-7721 cells changed after treatment with SPEF. Moreover, the adhesive force of low-dose SPEF-treated SMMC-7721 cells to endothelial cells markedly decreased compared to that of control cells. These results suggest that the suppressant effects of SPEF on the proliferation of SMMC-7721 cells appeared to be mediated, at least in part, through arresting cell cycle progression and altering the viscoelastic and adhesive properties of the cells, which provides a novel biorheological mechanism for the antitumor therapy of SPEF.
Conf Proc IEEE Eng Med Biol Soc. 2008;2008:1044-7.
Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field.
Yao C, Mi Y, Hu X, Li C, Sun C, Tang J, Wu X.
State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China.
This paper studies the apoptosis of human ovarian carcinoma cell Line (SKOV3) induced by the nanosecond pulsed electric field (10kV/cm, 100ns, 1 Hz) and its effect on intracellular calcium concentration ([Ca2+]i). These cells were doubly marked by Annexin V-FITC/PI, and the apoptosis rate was analyzed with flow cytometry. After AO/EB staining the morphological changes were observed under fluorescent microscope, and their ultrastructural changes were observed under scanning electron microscope (SEM). With Fluo-3/AM as calcium fluorescent marker, laser scanning confocal microscope (LSCM) was used to detect the effect of nsPEF on [Ca2+]i and the source of Ca2+. The results showed that the early apoptosis rate of the treatment group was (22.21+/-2.71)%, significantly higher than that of the control group (3.04+/-0.44)% (P<0.01). The typical features of apoptotic cell have been observed by fluorescent microscope and SEM. It is proved that nsPEF can induce apoptosis of SKOV3 cells and result in distinct increase in [Ca2+]i (P0.01), which was independent of extracellular calcium concentration (P>0.05). Since nsPEF can penetrate cell membrane due to its high frequency components, one of the mechanisms of nsPEF-induced apoptosis may be that activating intracellular calcium stores can increase the [Ca2+]i, and consequently, the apoptotic signal pathway can be induced.
Apoptosis. 2007 Sep;12(9):1721-31.
Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells.
Hall EH, Schoenbach KH, Beebe SJ.
Center for Pediatric Research, Children’s Hospital of the King’s Daughters, Department of Physiological Sciences, Eastern Virginia Medical School, PO Box 1980, Norfolk, VA 23501-1980, USA.
Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax.
Anticancer effects on leiomyosarcoma-bearing Wistar rats after electromagnetic radiation of resonant radiofrequencies.
Avdikos A, Karkabounas S, Metsios A, Kostoula O, Havelas K, Binolis J, Verginadis I, Hatziaivazis G, Simos I, Evangelou A.
Laboratory of Physiology, Unit of Environmental Physiology, Faculty of Medicine, University of Ioannina, Greece.
In the present study, the effects of a resonant low intensity static electromagnetic field (EMF), causing no thermal effects, on Wistar rats have been investigated. Sarcoma cell lines were isolated from leiomyosarcoma tumors induced in Wistar rats by the subcutaneous (s.c) injection of 3,4-benzopyrene. Furthermore, smooth muscle cells (SMC) were isolated from the aorta of Wistar rats and cultivated. Either leiomyosarcoma cells (LSC) or SMC were used to record a number of characteristic resonant radiofrequencies, in order to determine the specific electromagnetic fingerprint spectrum for each cell line. These spectra were used to compose an appropriate algorithm, which transforms the recorded radiofrequencies to emitted ones. The isolated LSC were cultured and then exposed to a resonant low intensity radiofrequency EMF (RF-EMF), at frequencies between 10 kHz to 120 kHz of the radiowave spectrum. The exposure lasted 45 consecutive minutes daily, for two consecutive days. Three months old female Wistar rats were inoculated with exposed and non-exposed to EMF LSC (4 x 10(6) LCS for animal). Inoculated with non-exposed to EMF cells animals were then randomly separated into three Groups. The first Group was sham exposed to the resonant EMF (control Group-CG), the second Group after the inoculation of LSC and appearance of a palpable tumor mass, was exposed to a non-resonant EMF radiation pattern, for 5 h per day till death of all animals (experimental control Group-ECG). The third Group of animals after inoculation of LSC and the appearance of a palpable tumor mass, was exposed to the resonant EMF radiation for 5 h per day, for a maximum of 60 days (experimental Group-I, EG-I). A fourth Group of animals was inoculated with LSC exposed to EMF irradiation and were not further exposed to irradiation (experimental Group-II, EG-II). Tumor induction was 100% in all Groups studied and all tumors were histologically identified as leiomyosarcomas. In the case of the EG-I, a number of tumors were completely regretted (final tumor induction: 66%). Both Groups of animals inoculated with exposed or non-exposed to the EMF LSC, (EG-I and EG-II, respectively) demonstrated a significant prolongation of the survival time and a lower tumor growth rate, in comparison to the control Group (CG) and the experimental control Group (ECG). However, the survival time of EG-I animals was found to be significantly longer and tumor growth rate significantly lower compared to EG-II animals. In conclusion, our results indicate a specific anticancer effect of resonant EMF irradiation. These results may possibly be attributed to (a) the duration of exposure of LSC and (b) the exposure of the entire animal to this irradiation.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Feb;24(1):230-4.
Biological effects and their applications in medicine of pulsed electric fields.
[Article in Chinese]
Huang H, Song G, Wang G, Sun C.
Key Laboratory for Biomnechanics & Tissue Engineering of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.
Pulsed electric fields can induce various kinds of biological effects that are essentially different from the normal electric fields, especially the interactions of Nanosecond Pulsed electric field (nsPEF) with cells. The biological effects of different pulsed electric fields on cell membranes, cytoplasmic matrixes, cell growth are introduced in this paper. Based on these effects, some applications of pulsed electric fields in cancer therapy, gene therapy, and delivery of drugs are reviewed in details.
Biochem Biophys Res Commun. 2006 May 5;343(2):351-60. Epub 2006 Mar 10.
Nanosecond pulsed electric fields cause melanomas to self-destruct.
Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, Kolb JF, Schoenbach KH.
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. email@example.com
We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.
Bioelectromagnetics. 2006 May;27(4):258-64.
Effect of millimeter wave irradiation on tumor metastasis.
Logani MK, Szabo I, Makar V, Bhanushali A, Alekseev S, Ziskin MC.
Richard J. Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, PA 19140, USA. firstname.lastname@example.org
One of the major side effects of chemotherapy in cancer treatment is that it can enhance tumor metastasis due to suppression of natural killer (NK) cell activity. The present study was undertaken to examine whether millimeter electromagnetic waves (MMWs) irradiation (42.2 GHz) can inhibit tumor metastasis enhanced by cyclophosphamide (CPA), an anticancer drug. MMWs were produced with a Russian-made YAV-1 generator. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm(2), respectively. Tumor metastasis was evaluated in C57BL/6 mice, an experimental murine model commonly used for metastatic melanoma. The animals were divided into 5 groups, 10 animals per group. The first group was not given any treatment. The second group was irradiated on the nasal area with MMWs for 30 min. The third group served as a sham control for group 2. The fourth group was given CPA (150 mg/kg body weight, ip) before irradiation. The fifth group served as a sham control for group 4. On day 2, all animals were injected, through a tail vein, with B16F10 melanoma cells, a tumor cell line syngeneic to C57BL/6 mice. Tumor colonies in lungs were counted 2 weeks following inoculation. CPA caused a marked enhancement in tumor metastases (fivefold), which was significantly reduced when CPA-treated animals were irradiated with MMWs. Millimeter waves also increased NK cell activity suppressed by CPA, suggesting that a reduction in tumor metastasis by MMWs is mediated through activation of NK cells.
Bioelectromagnetics. 2006 Apr;27(3):226-32.
Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi’s sarcoma-associated herpes virus in BCBL-1 cells.
Pica F, Serafino A, Divizia M, Donia D, Fraschetti M, Sinibaldi-Salimei P, Giganti MG, Volpi A.
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy.
Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P < .01), but not with percentage of gp K8.1A/B-positive cells or of cells containing virions. Viral progeny produced under ELF-EMF exposure consisted mainly of defective viral particles.
Conf Proc IEEE Eng Med Biol Soc. 2006;1:6370-2.
Outlook for the use of focused shock waves and pulsed electric fields in the complex treatment of malignant neoplasms.
Garilevich BA, Andrianov YV, Olefir YV, Zubkov AD, Rotov AE.
Central Air Force Clinical Hosp., Moscow, Russia.
The experimental studies the synchronous action of electric field microsecond range with amplitude within the range of 1-7 kV/sm and shock waves with pressure before 100 MPa on cells membrane permeability of the mouse’s ascitic tumors in vitro have shown the intensification the efficiency of the forming the irreversible pores under synchronous action. Thereby, enabling the electric field in the compression phase of shock wave pulse which can essentially reduce the electric field intensity required for breakdown cell membrane. In usual condition at amplitude of electric field, specified above, electric breakdown membrane carries basically reversible nature. At the same time in the pressure field tension phase of shock-wave pulse reversible pores, created by electric field, can grow before sizes, under which wholeness membrane is not restored. Under simultaneous action on cellular suspension the shock wave and electric field with moderate intensity cells survival is reduced in 5 once in contrast with occuring at different time’s action, and in 10 once in contrast with checking. The most sensitive to influence by under study fields are cells in phase of the syntheses DNA, preparation to fission and in phase of the mitosis. Thereby, continuation of the studies on use synchronous action shock waves and pulsed electric fields in complex treatment of the tumors introduces perspectiv
Bioelectromagnetics. 2006 Jan;27(1):64-72.
Effects of pulsed magnetic stimulation on tumor development and immune functions in mice.
Yamaguchi S, Ogiue-Ikeda M, Sekino M, Ueno S.
Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Japan.
We investigated the effects of pulsed magnetic stimulation on tumor development processes and immune functions in mice. A circular coil (inner diameter = 15 mm, outer diameter = 75 mm) was used in the experiments. Stimulus conditions were pulse width = 238 micros, peak magnetic field = 0.25 T (at the center of the coil), frequency = 25 pulses/s, 1,000 pulses/sample/day and magnetically induced eddy currents in mice = 0.79-1.54 A/m(2). In an animal study, B16-BL6 melanoma model mice were exposed to the pulsed magnetic stimulation for 16 days from the day of injection of cancer cells. A tumor growth study revealed a significant tumor weight decrease in the stimulated group (54% of the sham group). In a cellular study, B16-BL6 cells were also exposed to the magnetic field (1,000 pulses/sample, and eddy currents at the bottom of the dish = 2.36-2.90 A/m(2)); however, the magnetically induced eddy currents had no effect on cell viabilities. Cytokine production in mouse spleens was measured to analyze the immunomodulatory effect after the pulsed magnetic stimulation. tumor necrosis factor (TNF-alpha) production in mouse spleens was significantly activated after the exposure of the stimulus condition described above. These results showed the first evidence of the anti-tumor effect and immunomodulatory effects brought about by the application of repetitive magnetic stimulation and also suggested the possible relationship between anti-tumor effects and the increase of TNF-alpha levels caused by pulsed magnetic stimulation.
Clin Cancer Res. 2005 Oct 1;11(19 Pt 2):7093s-7103s.
Application of high amplitude alternating magnetic fields for heat induction of nanoparticles localized in cancer.
Ivkov R, DeNardo SJ, Daum W, Foreman AR, Goldstein RC, Nemkov VS, DeNardo GL.
Triton BioSystems, Inc., Chelmsford, Massachusetts 01824, USA. email@example.com
OBJECTIVE: Magnetic nanoparticles conjugated to a monoclonal antibody can be i.v. injected to target cancer tissue and will rapidly heat when activated by an external alternating magnetic field (AMF). The result is necrosis of the microenvironment provided the concentration of particles and AMF amplitude are sufficient. High-amplitude AMF causes nonspecific heating in tissues through induced eddy currents, which must be minimized. In this study, application of high-amplitude, confined, pulsed AMF to a mouse model is explored with the goal to provide data for a concomitant efficacy study of heating i.v. injected magnetic nanoparticles.
METHODS: Thirty-seven female BALB/c athymic nude mice (5-8 weeks) were exposed to an AMF with frequency of 153 kHz, and amplitude (400-1,300 Oe), duration (1-20 minutes), duty (15-100%), and pulse ON time (2-1,200 seconds). Mice were placed in a water-cooled four-turn helical induction coil. Two additional mice, used as controls, were placed in the coil but received no AMF exposure. Tissue and core temperatures as the response were measured in situ and recorded at 1-second intervals.
RESULTS: No adverse effects were observed for AMF amplitudes of < or = 700 Oe, even at continuous power application (100% duty) for up to 20 minutes. Mice exposed to AMF amplitudes in excess of 950 Oe experienced morbidity and injury when the duty exceeded 50%.
CONCLUSION: High-amplitude AMF (up to 1,300 Oe) was well tolerated provided the duty was adjusted to dissipate heat. Results presented suggest that further tissue temperature regulation can be achieved with suitable variations of pulse width for a given amplitude and duty combination. These results suggest that it is possible to apply high-amplitude AMF (> 500 Oe) with pulsing for a time sufficient to treat cancer tissue in which magnetic nanoparticles have been embedded.
Anticancer Res. 2005 Mar-Apr;25(2A):1023-8.
Frequency and irradiation time-dependant antiproliferative effect of low-power millimeter waves on RPMI 7932 human melanoma cell line.
Beneduci A, Chidichimo G, De Rose R, Filippelli L, Straface SV, Venuta S.
Department of Chemistry, University of Calabria, 87036 Arcavacata di Rende (CS), Italy. firstname.lastname@example.org
The biological effects produced by low power millimeter waves (MMW) were studied on the RPMI 7932 human melanoma cell line. Three different frequency-type irradiation modes were used: the 53.57-78.33 GHz wide-band frequency range, the 51.05 GHz and the 65.00 GHz monochromatic frequencies. In all three irradiation conditions, the radiation energy was low enough not to increase the temperature of the cellular samples. Three hours of radiation treatment, applied every day to the melanoma cell samples, were performed at each frequency exposure condition. The wide-band irradiation treatment effectively inhibited cell growth, while both the monochromatic irradiation treatments did not affect the growth trend of RPMI 7932 cells. A light microscopy analysis revealed that the low-intensity wide-band millimeter radiation induced significant morphological alterations on these cells. Furthermore, a histochemical study revealed the low proliferative state of the irradiated cells. This work provides further evidence of the antiproliferative effects on tumor cells induced by low power MMW in the 50-80 GHz frequency range of the electromagnetic spectrum.
Bioelectromagnetics. 2005 Jan;26(1):10-9.
Effect of millimeter waves on natural killer cell activation.
Makar VR, Logani MK, Bhanushali A, Kataoka M, Ziskin MC.
Richard J Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions.
Bioelectromagnetics. 2004 Oct;25(7):516-23.
Combined millimeter wave and cyclophosphamide therapy of an experimental murine melanoma.
Logani MK, Bhanushali A, Anga A, Majmundar A, Szabo I, Ziskin MC.
Richard J. Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
The objective of the present studies was to investigate whether millimeter wave (MMW) therapy can increase the efficacy of cyclophosphamide (CPA), a commonly used anti-cancer drug. The effect of combined MMW-CPA treatment on melanoma growth was compared to CPA treatment alone in a murine model. MMWs were produced with a Russian made YAV-1 generator. The device produced 42.2 +/- 0.2 GHz modulated wave radiation through a 10 x 20 mm rectangular output horn. The animals, SKH-1 hairless female mice, were irradiated on the nasal area. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm2, respectively. The maximum skin surface temperature elevation measured at the end of 30 min irradiation was 1.5 degrees C. B16F10 melanoma cells (0.2 x 10(6)) were implanted subcutaneously into the left flank of mice on day 1 of the experiment. On days 4-8, CPA was administered intraperitoneally (30 mg/kg/day). MMW irradiation was applied concurrently with, prior to or following CPA administration. A significant reduction (P < .05) in tumor growth was observed with CPA treatment, but MMW irradiation did not provide additional therapeutic benefit as compared to CPA alone. Similar results were obtained when MMW irradiation was applied both prior to and following CPA treatment.
Biofizika. 2004 May-Jun;49(3):545-50.
A comparison of the effects of millimeter and centimeter waves on tumor necrosis factor production in mouse cells.
[Article in Russian]
Sinotova OA, Novoselova EG, Glushkova OV, Fesenko EE.
The effects of millimeter (40 GHz) and centimeter (8.15-18.00 GHz) low-intensity waves on the production of tumor necrosis factor (TNE) in macrophages and lymphocytes from exposed mice as well as in exposed isolated cells were compared. It was found that the dynamics of TNF secretory activity of cells varies depending on the frequency and duration of exposure. The application of millimeter waves induced a nonmonotonous course of the dose-effect curve for TNF changes in macrophages and splenocytes. Alternately, a stimulation and a decrease in TNF production were observed following the application of millimeter waves. On the contrary, centimeter waves provoked an activation in cytokine production. It is proposed that, in contrast to millimeter waves, the single application of centimeter waves to animals (within 2 to 96 h) or isolated cells (within 0.5 to 2.5 h) induced a much more substantial stimulation of immunity.
Bioelectromagnetics. 2004 Oct;25(7):503-7.
Differences in lethality between cancer cells and human lymphocytes caused by LF-electromagnetic fields.
Radeva M, Berg H.
Labor Bioelectrochemistry (Campus Beutenberg, Jena) of the Saxonian Academy of Sciences, Leipzig, Germany.
The lethal response of cultured cancer cells lines K-562, U-937, DG-75, and HL-60 were measured directly after a 4 h exposure to a pulsating electromagnetic field (PEMF, sinusoidal wave form, 35 mT peak, 50 Hz) [Traitcheva et al. (2003): Bioelectromagnetics 24:148-158] and 24 h later, to determine the post-exposure effect. The results were found to depend on the medium, pH value, conductivity, and temperature. From these experiments, suitable conditions were chosen to compare the vitality between K-562 cells and normal human lymphocytes after PEMF treatment and photodynamic action. Both agents enhance necrosis synergistically for diseased as well as for healthy cells, but the lymphocytes are more resistant. The efficacy of PEMF on the destruction of cancer cells is further increased by heating (hyperthermia) of the suspension up to 44 degrees C or by lowering the pH-value (hyperacidity) to pH 6.4. Similar apoptosis and necrosis can be obtained using moderate magnetic fields (B < or = 15 mT 50/60 Hz), but this requires longer treatment of at least over a week. PEMF application combined with anticancer drugs and photodynamic therapy will be very effective.
Bioelectromagnetics. 2004 Sep;25(6):466-73.
Millimeter wave-induced suppression of B16 F10 melanoma growth in mice: involvement of endogenous opioids.
Radzievsky AA, Gordiienko OV, Szabo I, Alekseev SI, Ziskin MC.
Center for Biomedical Physics, Temple University Medical School, Philadelphia, Pennsylvania 19140, USA. email@example.com
Millimeter wave treatment (MMWT) is widely used in Eastern European countries, but is virtually unknown in Western medicine. Among reported MMWT effects is suppression of tumor growth. The main aim of the present “blind” and dosimetrically controlled experiments was to evaluate quantitatively the ability of MMWT to influence tumor growth and to assess whether endogenous opioids are involved. The murine experimental model of B16 F10 melanoma subcutaneous growth was used. MMWT characteristics were: frequency, 61.22 GHz; average incident power density, 13.3 x 10(-3) W/cm2; single exposure duration, 15 min; and exposure area, nose. Naloxone (1 mg/kg, intraperitoneally, 30 min prior to MMWT) was used as a nonspecific blocker of opioid receptors. Five daily MMW exposures, if applied starting at the fifth day following B16 melanoma cell injection, suppressed subcutaneous tumor growth. Pretreatment with naloxone completely abolished the MMWT-induced suppression of melanoma growth. The same course of 5 MMW treatments, if started on day 1 or day 10 following tumor inoculations, was ineffective. We concluded that MMWT has an anticancer therapeutic potential and that endogenous opioids are involved in MMWT-induced suppression of melanoma growth in mice. However, appropriate indications and contraindications have to be developed experimentally before recommending MMWT for clinical usage.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Aug;21(4):546-8.
Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle.
[Article in Chinese]
Yao C, Sun C, Mi Y, Xiong L, Hu L, Hu Y.
Key Lab of High Voltage Engineering and Electrical New Technology, Ministry of Education, Chongqing University, Chongqing 400044, China.
To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage). Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured. The DNA contents and cell cycle were analyzed by flow cytometry (FCM). Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability. With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident. The cells exposed to SPEFs grew slower than the control. The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups. It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase. There was a significant difference in cell cycle between treated group and control group (P<0.01). Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.
|Physiol Meas. 2004 Aug;25(4):1077-93.|
Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.
Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH.
Center for Pediatric Research, Eastern Virginia Medical School, Children’s Hospital for The King’s Daughters, Norfolk, VA, USA. firstname.lastname@example.org
These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.
|Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Jun;21(3):433-5.|
Effect of steep pulsed electric fields on survival of tumour-bearing mice.
[Article in Chinese]
Yao C, Sun C, Xiong L, Mi Y, Liao R, Hu L, Hu Y.
College of Electrical Engineering, Chongqing University, Chongqing, 400044, China.
To investigate the lethal effect of steep pulsed electric fields (SPEFs) on cancer cells and the life-prolonging effect of SPEFs on the survival of tumour-bearing mice, this study was carried out with the use of SPEFs to treat 40 BALB/C mice inoculated by cervical cancer. The lethal effect on cancer cells and the life-prolonging effect on tumour-bearing mice were examined and compared between the experiment group and control group. The survival periods of the experiment group and control group were 52.05 days and 33.03 days, respectively. There was a significant difference in survival curve between the two groups. The results confirmed the inhibitiory effect and lethal effect of SPEFs on cancer cells. SPEFs can prolong the survival period of tumour-bearing mice.
|Ann Biomed Eng. 2003 Jan;31(1):80-90.|
Viability of cancer cells exposed to pulsed electric fields: the role of pulse charge.
Krassowska W, Nanda GS, Austin MB, Dev SB, Rabussay DP.
Department of Biomedical Engineering, Duke University, Durham, NC 27708-0281, USA. email@example.com
The goal of this study was to collect a comprehensive set of data that related lethal effects of electric fields to the duration of the pulse. Electric pulses of different strengths and durations were applied to a suspension of HEp-2 cells (epidermoid carcinoma of the human larynx) using a six-needle electrode array connected through an autoswitcher to a square wave generator. Pulse durations varied from 50 micros to 16 ms and the ranges of electric field were adjusted for each duration to capture cell viabilities between 0% and 100%. After pulsation, cells were incubated for 44 h at 37 degrees C, and their viability was measured spectrophotometrically using an XTT assay. For each pulse duration (d), viability data were used to determine the electric field that killed half of the cells (E50). When plotted on logarithmic axes, E50 vs. d was a straight line, leading to a hyperbolic relationship: E50=const/d. This relationship suggests that the total charge delivered by the pulse is the decisive factor in killing HEp-2 cells.
|Vopr Onkol. 2003;49(6):748-51.|
Experience with turbulent magnetic field as a component of breast cancer therapy.
[Article in Russian]
Letiagin VP, Protchenko NV, Rybakov IuL, Dobrynin IaV.
N.N. Blokhin Center for Oncology Research, Russian Academy of Medical Sciences, Zdorovje Research Center, Moscow.
No adverse side-effects were reported in an investigation of the antitumor effect of turbulent magnetic field (TMF) carried out as a component of preoperative chemoradiotherapy for breast cancer at the Center’s Clinic. The study group included 114 patients with locally advanced tumors(T3, N1-N3, M0). According to the clinical, roentgenological and histological evidence on the end-results, the procedure was highly effective. Also, it was followed by shorter and less extensive postoperative lymphorrhea.
Bioelectromagnetics. 2003 Feb;24(2):148-50.
ELF fields and photooxidation yielding lethal effects on cancer cells.
Traitcheva N, Angelova P, Radeva M, Berg H.
Laboratory of Bioelectrochemistry, Institute of Virology, FSU, Jena, Germany.
The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.
Technol Cancer Res Treat. 2002 Feb;1(1):71-82.
Enhancing the effectiveness of drug-based cancer therapy by electroporation (electropermeabilization).
Rabussay DP, Nanda GS, Goldfarb PM.
Genetronics, Inc., 11199 Sorrento Valley Road, San Diego CA 92121, USA. firstname.lastname@example.org
Many conventional chemotherapeutic drugs, as well as DNA for cancer gene therapy, require efficient access to the cell interior to be effective. The cell membrane is a formidable barrier to many of these drugs, including therapeutic DNA constructs. Electropermeabilization (EP, often used synonymously with “electroporation”) has become a useful method to temporarily increase the permeability of the cell membrane, allowing a broad variety of molecules efficient access to the cell interior. EP is achieved by the application of short electrical pulses of relatively high local field strength to the target tissue of choice. In cancer therapy, EP can be applied in vivo directly to the tumor to be treated, in order to enhance intracellular uptake of drugs or DNA. Alternatively, EP can be used to deliver DNA into cells of healthy tissue to achieve longer-lasting expression of cancer-suppressing genes. In addition, EP has been used in ex vivo therapeutic approaches for the transfection of a variety of cells in suspension. In this paper, we communicate results related to the development of a treatment for squamous cell carcinomas of the head and neck, using electropermeabilization to deliver the drug bleomycin in vivo directly into the tumor cells. This drug, which is not particularly effective as a conventional therapeutic, becomes highly potent when the intracellular concentration is enhanced by EP treatment. In animal model experiments we found a drug dose of 1 U/cm(3) tumor tissue (delivered in 0.25 mL of an aqueous solution/cm3 tumor tissue) and an electrical field strength of 750 V/cm or higher to be optimal for the treatment of human squamous cell tumors grown subcutaneously in mice. Within 24-48 hours, the majority of tumor cells are rapidly destroyed by this bleomycin-electroporation therapy (B-EPT). This raises the concern that healthy tissue may be similarly affected. In studies with large animals we showed that normal muscle and skin tissue, normal tissue surrounding major blood vessels and nerves, as well as healthy blood vessels and nerves themselves, are much less affected than tumor tissue. Normal tissues did show acute, focal, and transitory effects after treatment, but these effects are relatively minor under standard treatment conditions. The severity of these effects increases with the number of electric pulse cycles and applied voltage. The observed histological changes resolved 20 to 40 days after treatment or sooner, even after excessive EP treatment. Thus, B-EPT is distinct from other ablative therapies, such as thermal, cryo, or photodynamic ablation, which equally affect healthy and tumor tissue. In comparison to surgical or radiation therapy, B-EPT also has potential as a tissue-sparing and function-preserving therapy. In clinical studies with over 50 late stage head and neck cancer patients, objective tumor response rates of 55-58%, and complete tumor response rates of 19-30% have been achieved.
Bioelectromagnetics. 2002 Dec;23(8):578-85.
Influence of 1 and 25 Hz, 1.5 mT magnetic fields on antitumor drug potency in a human adenocarcinoma cell line.
Ruiz-Gómez MJ, de la Peña L, Prieto-Barcia MI, Pastor JM, Gil L, Martínez-Morillo M.
Laboratory of Radiobiology, Department of Radiology and Physical Medicine, Faculty of Medicine, University of Málaga, Teatinos, Málaga, Spain.
The resistance of tumor cells to antineoplastic agents is a major obstacle during cancer chemotherapy. Many authors have observed that some exposure protocols to pulsed electromagnetic fields (PEMF) can alter the efficacy of anticancer drugs; nevertheless, the observations are not clear. We have evaluated whether a group of PEMF pulses (1.5 mT peak, repeated at 1 and 25 Hz) produces alterations of drug potency on a multidrug resistant human colon adenocarcinoma (HCA) cell line, HCA-2/1(cch). The experiments were performed including (a) exposures to drug and PEMF exposure for 1 h at the same time, (b) drug exposure for 1 h, and then exposure to PEMF for the next 2 days (2 h/day). Drugs used were vincristine (VCR), mitomycin C (MMC), and cisplatin. Cell viability was measured by the neutral red stain cytotoxicity test. The results obtained were: (a) The 1 Hz PEMF increased VCR cytotoxicity (P < 0.01), exhibiting 6.1% of survival at 47.5 microg/ml, the highest dose for which sham exposed groups showed a 19.8% of survival. For MMC at 47.5 microg/ml, the % of survival changed significantly from 19.2% in sham exposed groups to 5.3% using 25 Hz (P < 0.001). Cisplatin showed a significant reduction in the % of survival (44.2-39.1%, P < 0.05) at 25 Hz and 47.5 microg/ml, and (b) Minor significant alterations were observed after nonsimultaneous exposure of cells to PEMF and drug. The data indicate that PEMF can induce modulation of cytostatic agents in HCA-2/1(cch), with an increased effect when PEMF was applied at the same time as the drug. The type of drug, dose, frequency, and duration of PEMF exposure could influence this modulation.
Biofizika. 2002 Mar-Apr;47(2):376-81.
Immunomodulating effect of electromagnetic waves on production of tumor necrosis factor in mice with various rates of neoplasm growth.
[Article in Russian]
Glushkova OV, Novoselova EG, Sinotova OA, Vrublevskaia VV, Fesenko EE.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.
The effects of low-density centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 14 days; MW) on tumor necrosis factor production in macrophages of mice with different growth rate of a cancer solid model produced after hypodermic injection of Ehrlich carcinoma ascites cells into hind legs were studied. After irradiation, an increase in the concentration of tumor necrosis factor in immunocompetent cells of healthy and, specially, of tumor-bearing animals was observed; and the effect of stimulation was higher upon exposure of mice carrying rapidly growing tumors. We suggest that the significant immunomodulating effect of low-density microwaves can be utilized for tumor growth suppression.
Biofizika. 2001 Jan-Feb;46(1):131-5.
Effect of centimeter m
Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.
Biomedical Physics Unit, Department of Physics, Wuhan University, Wuhan, 430072, China.
Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.
icrowaves and the combined magnetic field on the tumor necrosis factor production in cells of mice with experimental tumors.
[Article in Russian]
Novoselova EG, Oga? VB, Sorokina OV, Novikov VV, Fesenko EE.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.
The effect of fractionated exposure to low-intensity microwaves (8.15-18 GHz, 1 microW/cm2, 1.5 h daily for 7 days) and combined weak magnetic field (constant 65 1 microT; alternating–100 nT, 3-10 Hz) on the production of tumor necrosis factor in macrophages of mice with experimental solid tumors produced by transplantation of Ehrlich ascites carcinoma was studied. It was found that exposure of mice to both microwaves and magnetic field enhanced the adaptive response of the organism to the onset of tumor growth: the production of tumor necrosis factor in peritoneal macrophages of tumor-bearing mice was higher than in unexposed mice.
|J Photochem Photobiol B. 2001 Nov 1;64(1):21-6.|
Photodynamic effect on cancer cells influenced by electromagnetic fields.
Pang L, Baciu C, Traitcheva N, Berg H.
Institute of Physics, Nankai University, Nankai, PR China.
The synergism of low-frequency electromagnetic field treatment and photodynamic effect on killing of human cancer cells is presented. The weak pulsating electromagnetic field (PEMF) generated by Helmholtz coils in the mT range influences the permeability of cell membranes for photosensitizers. Several types of sensitizers were excited by visible light during incorporation without and with two kinds of PEMF treatment. In the first part suitable photosensitizers were selected in the absorption range between 400 and 700 nm against human myeloid leukaemia K562 and human histiocytic lymphoma U937 cells by treatment of PEMF consisting of rectangular pulse groups. In the second part amplitude and frequency dependencies were measured using sinuous PEMF and white light with the result that after 12 min the PEMF treatment enhanced photodynamic effectivity by more than 40% over the control value. Taking into account the influence of many parameters, an additional optimization will be possible by photodynamic PEMF synergism for an increased drug delivery in general.
Bioelectromagnetics. 2001 Oct;22(7):503-10.
Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells.
Pessina GP, Aldinucci C, Palmi M, Sgaragli G, Benocci A, Meini A, Pessina F.
Institute of General Physiology and Nutritional Science, University of Siena, Italy. email@example.com
Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure.
Cell Mol Biol (Noisy-le-grand). 2001;47 Online Pub:OL115-7.
Inhibition of proliferation of human lymphoma cells U937 by a 50 Hz electromagnetic field.
Glück B, Güntzschel V, Berg H.
Laboratory Cell Culture, Institute of Virology, Friedrich-Schiller-University Jena, Germany. firstname.lastname@example.org
Weak pulsating electromagnetically induced fields (PEMF) by Helmholtz coils changes cell metabolism, if cells are treated with a certain range of frequency and amplitude. The influence on proliferation of human histiocytic lymphoma cells U937 has been studied applying a sinusoidal 50 Hz field with amplitudes of the flux density B = 0.3 to 4.7 mT for 4 days. No difference between experiment and control was found in the region 0.3 and 2 mT. However, stronger fields (> or =2.5 mT) inhibit cell division. Fields > or =3.5 mT treatment kill > or =80% of the cell number at the beginning (1.5 x 10(5)/ml). This effect may be an electromagnetocally induced cell death as the first step for a non-invasive application on cell proliferation process.
Laryngoscope. 2001 Jan;111(1):52-6.
Electroporation therapy for head and neck cancer including carotid artery involvement.
Allegretti JP, Panje WR.
Department of Otolaryngology, Rush-Presbyterian-St Luke’s Medical Center, Rush Medical College, Chicago, Illinois 60612, USA.
OBJECTIVES: Electroporation therapy with intralesional bleomycin (EPT) is a novel, technically simple outpatient technique in which high-voltage electric impulses delivered into a neoplasm transiently increase cell membrane permeability to large molecules, including cytotoxic agents, causing localized progressive necrosis. Unlike many laser ablation methods, EPT can treat bulky tumors (>2 cm) with complete penetration. Our recent publication confirms an excellent response rate in the use of EPT in a clinical trial. STUDY
DESIGN, PATIENTS, AND METHODS: Following our initial prospective study report in 1998, we have followed our entire initial cohort (10 patients) of patients with head and neck cancer beyond 24-months follow-up. Additionally, we have used this approach to treat four additional patients (total: 9 males/5 females) with upper aerodigestive tract squamous cell carcinoma, including three with internal carotid artery (ICA) involvement up to or within the skull base. Two patients underwent preoperative balloon test occlusion with cerebral perfusion studies followed by carotid embolization. EPT was then done safely at least 2 weeks later to avoid the temporary hypercoagulable state.
RESULTS: Within the overall cohort (14 patients) 6 patients had a complete response, 6 had a partial response, and 2 did not respond (overall 85.7% response rate). Both patients with ICA involvement had a partial or complete response to treatment; neither patient had a hemorrhagic or neurologic complication. Overall, 13 of the 14 patients were treated for persistent or recurrent head and neck cancer. Two of the four patients with early recurrent stage tumors had no evidence of recurrence after EPT with an average follow-up of 31.5 months. The overall early stage tumor group had four complete responders out of five (80%). On the contrary, only 2 of 9 patients with advanced recurrent stage tumors were disease-free at 18 months. Morbidity was low for early stage tumors, but higher for advanced tumors with complications, including poor wound healing, dysphagia, and osteomyelitis. There were no treatment-related deaths.
CONCLUSION: We found EPT to be safe and efficacious in patients with head and neck cancer, even with internal carotid artery involvement. Patients with early stage recurrences have the potential for prolonged survival beyond 2 years without the morbidity of surgery and radiation or toxicity of systemic chemotherapy. Because of its superb access qualities even for bulky tumors, EPT is a potential method of delivery for other tumoricidal agents such as in genetic-altering schemes.
|Vopr Onkol. 2000;46(4):469-72.|
Use of artificial magnetic field for rehabilitation of children with malignant tumors.
[Article in Russian]
Kiselev AV, Grushina TI.
N.N. Blokhin Center for Oncology Research, Russian Academy of Medical Sciences, Moscow.
Local complications of standard intravenous injections for chemotherapy and due to error of administration were compared in 400 patients (200 of them children) and general wound pathologies described. Treatment for wounds included two modalities: standard medication and alternating or pulsating magnetic field. Magnetic therapy proved highly effective: wound healing was 3-3.5 times faster while duration of treatment–2-3 times shorter than in standard procedure. Clinically-verified partial adhesion-related intestinal obstruction was eliminated by magnetic procedure in 18 children after combined treatment for lymphosarcoma involving the ileum.
Bioelectromagnetics. 2000 Feb;21(2):112-21.
Effects of PEMF on a murine osteosarcoma cell line: drug-resistant (P-glycoprotein-positive) and non-resistant cells.
Miyagi N, Sato K, Rong Y, Yamamura S, Katagiri H, Kobayashi K, Iwata H.
Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Japan.
After pulsed exposure of Dunn osteosarcoma cells (nonresistant cells) to Adriamycin (ADR) at increasing concentrations and single-cell cloning of surviving cells, ADR-resistant cells were obtained. These resistant cells expressed P-glycoprotein and had resistance more than 10 times that of their nonresistant parent cells. Compared to the nonresistant cells not exposed to pulsing electromagnetic fields (PEMF) in ADR-free medium, their growth rates at ADR concentrations of 0.01 and 0.02 micrograms/ml, which were below IC50, were 83.0% and 61.8%, respectively. On the other hand, in the nonresistant cells exposed to PEMF (repetition frequency, 10 Hz; rise time, 25 microsec, peak magnetic field intensity, 0.4-0.8 mT), the growth rate was 111.9% in ADR-free medium, 95.5% at an ADR concentration of 0.01 micrograms/ml, and 92.2% at an ADR concentration of 0.02 micrograms/ml. This promotion of growth by PEMF is considered to be a result of mobilization of cells in the non-proliferative period of the cell cycle due to exposure to PEMF. However, at ADR concentrations above the IC50, the growth rate tended to decrease in the cells not exposed to PEMF. This may be caused by an increase in cells sensitive to ADR resulting from mobilization of cells in the non-proliferative period to the cell cycle. The growth rate in the resistant cells exposed to PEMF was significantly lower than that in the non-exposed resistant cells at all ADR concentrations, including ADR-free culture (P</=0.0114). Therefore, this study suggests that PEMF promotes the growth of undifferentiated cells but progressively suppresses the growth of more differentiated cells, i.e., PEMF controls cell growth depending on the degree of cell differentiation. This study also shows the potentiality of PEMF as an adjunctive treatment method for malignant tumors
J Physiol Biochem. 1999 Jun;55(2):79-83.
Growth modification of human colon adenocarcinoma cells exposed to a low-frequency electromagnetic field.
Ruiz Gómez MJ, Pastor Vega JM, de la Peña L, Gil Carmona L, Martínez Morillo M.
Departamento de Radiología y Medicina Física, Facultad de Medicina, Universidad de Málaga, Teatinos, Spain. email@example.com
The influence of variable low-intensity, low-frequency electromagnetic fields on culture cells is investigated. Human colon adenocarcinoma cells were exposed to a rectangular and variable magnetic field (1 and 25 Hz; 1.5 mT peak). Cultures were exposed to a dose for 15 and 360 minutes, and after 24 hours incubation, cell viability was measured with neutral red stain. The group treated for 15 minutes showed a statistically significant increase in cell growth with 1 Hz (p < 0.002) and 25 Hz (p < 0.003). In contrast, a significant decrease in cell growth was found in those cultures treated with 1 Hz for 360 minutes (p < 0.02). The effects reported could be influenced by the magnetic field frequency and the exposure time.
|Am J Physiol. 1997 May;272(5 Pt 2):R1677-83.|
Electrical fields enhance growth of cancer spheroids by reactive oxygen species and intracellular Ca2+.
Wartenberg M, Hescheler J, Sauer H.
Institute for Neurophysiology, University of Cologne, Germany.
A single electrical field pulse (500 V/m) with a duration of 60 s increased tumor outgrowth over a postpulse period of 24 h. RNA staining with acridine orange showed a rise in RNA content in pulsed spheroids, indicating stimulation of cell cycle activity. The electropulse induced an intracellular Ca2+ concentration ([Ca2+]i) transient that started approximately 40 s after the onset of the electrical field. Neither the presence of extracellular Ni2+ (0.5 mM) nor the absence of extracellular Ca2+ impeded the [Ca2+]i rise. It was, however, totally blocked by thapsigargin (1 microM), indicating that the initial Ca2+ response is due to Ca2+ release from intracellular stores. The [Ca2+]i transient was paralleled by an increase in reactive oxygen species (ROS), as revealed using 2′,7′-dichlorofluorescein diacetate as an indicator. The radical scavengers N-acetyl-L-cysteine (NAC)(20 mM) and dehydroascorbate (5 mM) inhibited both ROS production and the [Ca2+]i transient during electrical field treatment. The mitogenic activation was dependent on the rise in [Ca2+]i because inhibition of Ca2+ release during electrical field treatment by addition of either thapsigargin or NAC to the incubation medium abolished the observed effect. We conclude that a single, direct current electrical field pulse induces production of ROS, which in turn mediate Ca2+ release from intracellular stores and activate cell cycle activity in multicellular spheroids.
Anticancer Res. 1997 May-Jun;17(3C):2083-8.
Enhanced potency of daunorubicin against multidrug resistant subline KB-ChR-8-5-11 by a pulsed magnetic field.
Liang Y, Hannan CJ Jr, Chang BK, Schoenlein PV.
Department of Radiology, Research and Nuclear Medicine, Medical College of Georgia, Augusta 30912, USA.
Tumor cell resistance to many unrelated anticancer drugs is a major obstacle during cancer chemotherapy. One mechanism of drug resistance is thought to be due to the efflux of anticancer drugs caused by P-glycoprotein. In recent years, magnetic fields have been found to enhance the potency of anticancer drugs, with favorable modulation of cancer therapy. In this study, KB-ChR-8-5-11, a multidrug resistant (MDR) human carcinoma subline, was used as a model to evaluate the ability of pulsed magnetic fields (PMF) to modulate the potency of daunorubicin (DNR) in vivo and to determine the appropriate order of exposure to drugs and PMF using an in vitro cytotoxicity assay. Solenoid coils with a ramped pulse current source were used at 250 pulses per second for both in vivo and in vitro experiments. For the in vivo study, KB-ChR-8-5-11 cells were inoculated into thymic Balbc-nu/nu female mice. Treatment was begun when the average tumor volume reached 250-450 mm3. Treatment consisted of whole body exposure to PMF for one hour, followed immediately by intravenous (i.v.) injection of 8 mg/kg DNR designated as day 0, and repeated on days 7 and 14. Among the various groups, significant differences in the tumor volume were found between PMF + saline and PMF + DNR groups (p = 0.0107) at 39 days and 42 days (p = 0.0101). No mice died in the PMF alone group, and no toxicity attributable to PMF was found during the experimental period. For the in vitro studies, the sulforhodamine blue (SRB) cytotoxicity assay was used to determine the effect of the sequence which cells are exposed to PMF and/or DNR. Cells were exposed to PMF either before (pre-PMF) or after (post-PMF) drug was added. Results showed that the IC50 was significantly different between controls and pre-PMF + DNR groups (P = 0.0096, P = 0.0088). The IC50 of the post-PMF + DNR group was not found to be significantly different from control groups. Thus, the data in this report demonstrates that PMF enhanced the potency of DNR against KB-ChR-8-5-11 xenograft in vivo, while the efficacy of DNR was potentiated in vitro by PMF exposure only when PMF exposure occurred in the presence of drug. The data in vitro suggest that the mechanism by which PMFs modulate DNR’s potency may be by inhibition of the efflux pump, P-glycoprotein. Further work to determine conditions for maximum modulation of drug potency by PMFs is warranted.
Zhongguo Zhong Xi Yi Jie He Za Zhi. 1997 May;17(5):286-8.
Effect of acupoint irradiation with Q-wave millimeter microwave on peripheral white blood cells in post-operational treatment with chemotherapy in stomach and colorectal cancer patients.
[Article in Chinese]
Wu JG, Huang WZ, Wu BY.
Oncology Department of Second Ningde District Hospital, Fujian.
OBJECTIVE: To explore the biological effect of Q-wave millimeter microwave (QWMM).
METHODS: The QWMM was used to irradiate the acupoints Xuehai (Sp10) and Geshu (B17) in treating post-operational and chemotherapy treated stomach cancer and colorectal cancer patients. The effect of irradiation on chemotherapy affected peripheral white blood cells was observed. 62 cases (stomach cancer 42, colorectal cancer 20) in total were divided into two groups: group A, 21 cases (stomach cancer 15, colorectal cancer 6) the irradiation began 10 days after operation, and on the 16th day the chemotherapy combined with irradiation started. Group B had 41 cases (stomach cancer 27, colorectal cancer 14), in which the irradiation began immediately after the occurrence of chemotherapy induced peripheral WBC reduction, which persisted for at least 12 days.
RESULTS: The effective rate for the group A and B was 85.7% (18/21) and 73.2% (30/41) respectively. The total effective rate of the two groups was 77.4% (48/62). The effective rate of group A was significantly higher than that of group B, P < 0.01.
CONCLUSION: GWMM irradiation at acupoints could promote the hematopoietic function of bone marrow, and the irradiation performed 1 week before chemotherapy yielded even better protection on bone marrow.
Exposure to strong static magnetic field slows the growth of human cancer cells in vitro.
Raylman RR, Clavo AC, Wahl RL.
University of Michigan Medical Center, Department of Internal Medicine, Ann Arbor, USA.
Proposals to enhance the amount of radiation dose delivered to small tumors with radioimmunotherapy by constraining emitted electrons with very strong homogeneous static magnetic fields has renewed interest in the cellular effects of prolonged exposures to such fields. Past investigations have not studied the effects on tumor cell growth of lengthy exposures to very high magnetic fields. Three malignant human cell lines, HTB 63 (melanoma), HTB 77 IP3 (ovarian carcinoma), and CCL 86 (lymphoma: Raji cells), were exposed to a 7 Tesla uniform static magnetic field for 64 hours. Following exposure, the number of viable cells in each group was determined. In addition, multicycle flow cytometry was performed on all cell lines, and pulsed-field electrophoresis was performed solely on Raji cells to investigate changes in cell cycle patterns and the possibility of DNA fragmentation induced by the magnetic field. A 64 h exposure to the magnetic field produced a reduction in viable cell number in each of the three cell lines. Reductions of 19.04 +/- 7.32%, 22.06 +/- 6.19%, and 40.68 +/- 8.31% were measured for the melanoma, ovarian carcinoma, and lymphoma cell lines, respectively, vs. control groups not exposed to the magnetic field. Multicycle flow cytometry revealed that the cell cycle was largely unaltered. Pulsed-field electrophoresis analysis revealed no increase in DNA breaks related to magnetic field exposure. In conclusion, prolonged exposure to a very strong magnetic field appeared to inhibit the growth of three human tumor cell lines in vitro. The mechanism underlying this effect has not, as yet, been identified, although alteration of cell growth cycle and gross fragmentation of DNA have been excluded as possible contributory factors. Future investigations of this phenomenon may have a significant impact on the future understanding and treatment of cancer.
|J Cell Biochem. 1993 Apr;51(4):387-93.|
Beneficial effects of electromagnetic fields.
Bioelectric Research Center, Columbia University, Riverdale, New York 10463.
Selective control of cell function by applying specifically configured, weak, time-varying magnetic fields has added a new, exciting dimension to biology and medicine. Field parameters for therapeutic, pulsed electromagnetic field (PEMFs) were designed to induce voltages similar to those produced, normally, during dynamic mechanical deformation of connective tissues. As a result, a wide variety of challenging musculoskeletal disorders have been treated successfully over the past two decades. More than a quarter million patients with chronically ununited fractures have benefitted, worldwide, from this surgically non-invasive method, without risk, discomfort, or the high costs of operative repair. Many of the athermal bioresponses, at the cellular and subcellular levels, have been identified and found appropriate to correct or modify the pathologic processes for which PEMFs have been used. Not only is efficacy supported by these basic studies but by a number of double-blind trials. As understanding of mechanisms expands, specific requirements for field energetics are being defined and the range of treatable ills broadened. These include nerve regeneration, wound healing, graft behavior, diabetes, and myocardial and cerebral ischemia (heart attack and stroke), among other conditions. Preliminary data even suggest possible benefits in controlling malignancy.
|In Vivo. 1991 Jan-Feb;5(1):39-40.|
Effect of a 9 mT pulsed magnetic field on C3H/Bi female mice with mammary carcinoma. A comparison between the 12 Hz and 460 Hz frequencies.
Bellossi A, Desplaces A.
Laboratoire de Biophysique, Faculte de Medecine, Rennes, France.
In a previous experiment, the exposure of tumoral C3H/Bi female mice to a 9 mT, 460 Hz pulsed magnetic field led to an increase in the length of survival in the late period of the disease; this might be due to a hampered metastatic process. In the present study 27 controls and 52 exposed mice were treated with the same protocol (a 10-minute exposure, 3 non-consecutive days a week, from 2-3 weeks after the tumors appeared until death) but with a 12 Hz PMF. In this experiment the 12 Hz PMF appeared to increase length of survival times in the early period of the disease.
|Sov Med. 1991;(7):25-7.|
The assessment of the efficacy of the effect of a rotational magnetic field on the course of the tumor process in patients with generalized breast cancer.
[Article in Russian]
Bakhmutskii NG, Pyleva TA, Frolov VE, Sinitskii DA, Ripa IM.
The efficacy of rotational magnetic field generated by a “Magnitoturbotron” unit was evaluated in 51 women with advanced breast cancer. The effect resulted from an action on the patient’s body by modulated rotational magnetic field changing in cycles according to induction. A significant response was achieved in 27 of 51 patients. There was no hemopoiesis suppression, negative functional shifts. The unit is recommended for introduction in a combined treatment of breast cancer.
|Jpn J Cancer Res. 1990 Sep;81(9):956-61.|
Treatment of experimental tumors with a combination of a pulsing magnetic field and an antitumor drug.
Omote Y, Hosokawa M, Komatsumoto M, Namieno T, Nakajima S, Kubo Y, Kobayashi H.
Laboratory of Pathology, Hokkaido University School of Medicine, Sapporo.
We investigated the effects of a combination treatment involving a pulsing magnetic field (PMF) and an antitumor drug, mitomycin C (MMC), on two experimental tumors (fibrosarcoma KMT-17 and hepatocellular carcinoma KDH-8) in WKA rats, paying attention to possible potentiation of the therapeutic effect of the antitumor drug. PMF was obtained using a system generating a pulsed current in a solenoid coil. On day 7 after tumor implantation into the right thighs of rats, the region of the tumor was exposed to PMF (frequency 200 Hz, mean magnetic flux density 40 gauss) for 1 h immediately after iv injection of MMC at a dose of 1 mg/kg. Survival rates at day 90 of KMT-17 implanted rats were 0% (0/10) in the non-treated group, 34% (4/12) in the MMC-treated group, 47% (6/13) in the PMF-treated group and 77% (10/13) in the MMC/PMF combination group. The increase of life span (ILS) of KDH-8-implanted rats in the combination therapy group was significantly prolonged (%ILS 17.6%) compared with that in the MMC-treated (%ILS 3.4%) and PMF-treated (%ILS 7.6%) groups. By using cultured cells of the above two lines of tumor, the therapeutic effects of MMC and PMF were also determined from the cell colony-forming efficiency in soft agar. The colony-forming efficiencies of both cell lines were significantly suppressed in the combination therapy group compared with those in the other single therapy groups. The present results indicate that PMF exhibited a potentiation of the antitumor effect of mitomycin C.
Nippon Geka Gakkai Zasshi. 1988 Aug;89(8):1155-66.
An experimental attempt ot potentiate therapeutic effects of combined use of pulsing magnetic fields and antitumor agents.
[Article in Japanese]
First Department of Surgery, Asahikawa Medical College, Japan.
With a view to examining the possible clinical applicability of pulsing magnetic fields (PMF), we investigated the effects of weak, non-heat inducing, PMF on DNA synthesis and sensitivity of cancer cells to antitumor agents. Leukemic T-cells (Molt-4) and a pancreatic ductal adenocarcinoma (solid tumor) transplanted in a Syrian golden hamster were used for the in vitro experiment and in vivo experiment respectively. In order to evaluate the effects of PMF on the DNA synthesis of cancer cells and the incorporation of antitumor agent into cancer cells, cultured cells or solid tumor were exposed to PMF generated by a solenoid coil immediately after 3H-or 14C-thymidine and 3H-methotrexate administration respectively. Thymidine uptake was found to increase by exposure to PMF, as did also 3H-methotrexate uptake by leukemic T-cells. Following exposure to PMF immediately after administration of methotrexate or mitomycin C, antitumor activity in both cells was increased. From these results it appears that the incorporation of antitumor agents into the cells increases by eddy current stimulation induced by PMF, and that the cell cycle shifts from the non-proliferative to proliferative phase, resulting in increased antitumor activity.
|Anticancer Res. 1987 May-Jun;7(3 Pt B):391-3.|
Tumoricidal cells increased by pulsating magnetic field.
Malter M, Schriever G, Kuhnlein R, Suss R.
Repeated applications of pulsed magnetic fields (right-angle waves, 50 Hz = 135 Gauss, 2 Hz = 262 Gauss) significantly enhanced the number and the tumoricidal activity of nonparenchymal liver cells. The transplantable mouse leukemia L1210 used as a tumor model was not significantly influenced, either directly or during Cyclophosphamide treatment
|Vopr Onkol. 1980;26(1):28-34.|
Morphological criteria of lung cancer regression under the effect of magnetotherapy.
[Article in Russian]
Ogorodnikova LS, Gairabed’iants NG, Ratner ON, Chirvina ED, Sem LD.
The complex investigation (histological, histochemical, morphological, electrone microscopy) of lung cancerous tumors from 20 patients, subjected preoperatively to the action of magnetic fields enhancing the antitumor resistance by developing general nonspecific adaptation reactions: activation and training, has revealed a number of morphological changes which indicate a marked antitumor effect of magnetic fields. These changes were maximum manifest after 20-30 sessions. High-differentiated adenocarcinoma proved to be mostly sensitive to the magnetic field action.