Page | 003 BEMER Therapy Reduces Cancer Cell Radioresistance
By focusing on cells from solid tumors, we explored how the BEMER EMF pattern affects the metabolome in terms of glycolysis and tricarboxylic acid (TCA) cycles and the sensitivity to radiotherapy, chemotherapy and Cetuximab. To better address this question, we utilized a more physiological 3D laminin-rich extracellular matrix (lrECM)-based cell culture model. We found a significant radiosensitization of cancer cells by the BEMER therapy mechanisti- cally derived from higher levels of reactive oxygen species and increased numbers of DNA double strand breaks (DSBs).
Materials and Methods
Cell culture and irradiation
Human head and neck squamous carcinoma (HNSCC) cell line UTSCC15 was kindly pro- vided by R. Grenman (Turku University Central Hospital, Finland), human lung carcinoma cell line A549, human colorectal carcinoma cell line DLD1 and human pancreatic ductal ade- nocarcinoma cell line MiaPaca2 were purchased from American Tissue Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAA, Co ̈lbe, Germany) containing glutamax-I supplemented with 10% fetal calf serum (FCS; PAA) and 1% non-essen- tial amino acids (PAA) at 37 ̊C in a humidified atmosphere containing 8.5% CO2. In all experi- ments, asynchronously growing cells were used. Three (3D)-dimensional cell cultures were accomplished by imbedding cells in 0.5 mg/ml lrECM (MatrigelTM; BD, Heidelberg, Germany) [28–30]. Irradiation was performed at room temperature using single doses of 200 kV X-rays (Yxlon Y.TU 320; Yxlon; dose rate ~1.3 Gy/min at 20 mA) filtered with 0.5 mm Cu. The absorbed dose was measured using a Duplex dosimeter (PTW).
BEMER therapy
BEMER (Bio-Electro-Magnetic-Energy-Regulation) therapy uses a low-frequency pulsed mag- netic field [22,23] which was applied for 8 min, 1 h or 24 h. The detailed physical properties of this device are reviewed in the following patents: EP 0995463 A1, WO 2008025731 A1; WO 2011023634 A1 [31–33]. The electromagnetic field (EMF) with a pulse-duration of 30 ms and a pulse-frequency of 30 Hz was generated by a commercially available control unit B.
Box Classic (BEMER AG Int.; Fig 1A) with 10 different levels of magnetic field intensity (from 0 μT to 35 μT) and a mattress applicator (Fig 1B) with a flat coil system (Bio-Electromagnetic- Energy-Regulation, BEMER International AG, Triesen, Liechtenstein). The pulse generator is fed with a mains voltage of 230 V AC / 50 Hz. Based on the commercially available construc- tion, this mattress applicator was specifically designed for cell culture use with a maximum operating voltage of 12 V DC. Additionally, different signal intensities were used at level 1 (~2.7 μT), level 4 (~13 μT), level 7 (~23 μT) and level 10 (~35 μT). The signal is a sequence of individual pulses with a pulse width of approximately 33 milliseconds in the altitude of 3 to
35 μT within a predetermined time period of 18 to 22 seconds. The preferred exponential func- tion described in detail in EP 0995463 A1 is y = (x 3 • esin(x3)):c (with y as amplitude) [31]. The amplitudes of the single pulses correspond to an e-function and are then summarized as a group of pulses. As shown in fig 1C, BEMER-treated cells were placed within the labeled area above the flat coil on the mattress, and then stimulated with indicated intensities for 8 min, 1 h or 24 h. BEMER therapy was conducted at 37 ̊C in a humidified atmosphere containing 8.5% CO2 for pH 7.4. Control cells were sham-treated by placing them on the BEMER applicator for the respective time without applying the BEMER signal. BEMER signal intensity was measured using a 3D teslameter (PCE-G28, PCE, Germany) and cells were placed in the same area of the BEMER applicator for each treatment.
PLOS ONE | DOI:10.1371/journal.pone.0167931 December 13, 2016 3 / 19
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