Reagent and chemicals
DMEM (Dulbecco's Modified Eagle Medium)—Lonza, Fetal Bovine Serum—Lonza, Penicillin—Sigma Aldrich, Amphotericin B—Sigma Aldrich, Trypsin EDTA solution—Lonza, 0.4% Trypan Blue—Invitrogen, DMSO (dimethyl sulfoxide)—Sigma Aldrich, PBS (buffered saline solution)—Lonza, Baicalin—Sigma Aldrich, Baicalein—Sigma Aldrich, Protease inhibitor—Sigma Aldrich, Phosphatase inhibitor—Sigma Aldrich, TRIzol reagent—Invitrogen.
Incubator—Haraeus, Countess TM Automated Cell Counter—Invitrogen, Centrifuge—MPW 223e, Incubator—Incucell, Nano MN-913″—Maestrogen, DNA Engine Opticon™ System—MJ Research Inc, Multi-functional microplate reader—Victor.
Cell culture conditions
Amelanotic melanoma cells (C32 cell line) were obtained from ATCC (CRL-1585; Manassas, VA, USA) and routinely maintained in a DMEM medium, which was supplemented with 10% fetal bovine serum, penicillin (10,000 U/ml) and amphotericin B (0.25 mg/mL) at 37 °C in a 5% CO2 incubator (Heraeus).
Both the number of cells and their viability were monitored by cell counting in a Countess TM Automated Cell Counter (Invitrogen, USA) after staining with 0.4% trypan blue. The experiment was performed on cells that were in the logarithmic phase of growth under conditions of ≥ 98% viability as assessed by the trypan blue exclusion. For the experiments, the melanoma cells were used at three to five passages. The control melanoma cells and melanoma cells that had been treated with baicalin or baicalein were then subjected to a static magnetic field. The baicalin and baicalein concentration was selected with 0.1–250 µmol/L in a pilot study. The concentration 50 µmol/L was selected for the experiment because it was cytotoxic for the melanoma cells and non-cytotoxic for the skin fibroblasts (NHDF cell line). The addition of 50 µmol/L baicalin or baicalein to melanoma cells culture reduced cell viability by about 20% compared to the control . Melanoma cells were cultured in culture flasks. One million cells were introduced into each flask, and after 24 h incubation, they were treated with 50 µmol/L baicalin or baicalein and placed in test chambers emitting a static magnetic field with an induction of 0.7 T. The cultures were maintained in the test chambers at 37 °C in a 5% CO2 incubator (Heraeus) for 24 h. Next, the cells were washed with PBS and the cell numbers were determined by cell counting in a Countess TM Automated Cell Counter (Invitrogen, USA) after staining with 0.4% trypan blue.
Exposure of melanoma cells to static magnetic fields
To study the cells in a static magnetic field, magnetic chambers composed of permanent magnets and a ferromagnetic yoke were used (patent P-396639). The ferromagnetic yoke is the bottom and cover of the chamber and there is a window in the front wall of the chamber that is matched to the dimensions of the cell culture flask . A homogeneous distribution of the magnetic induction over the surface of the flask is conditioned by the structure of the test chamber. The static magnetic field is generated by neodymium magnets and the magnetic field intensity is proportional to the magnetic field strength. The chambers are constructed with the following materials: N42SH magnets, Br = 1.28–1.34 T, HcB ≥ 955 kA/m, HcJ ≥ 1512 kA/m, (BH)max = 310–342 kJ/m3, S235JR steel and a diamagnetic material. The maximum operating temperature of the chambers is 150 °C. A chamber with a field induction of 0.7 T was used for the tests, which was checked with a gauss meter before each experiment. The control chamber is made of steel instead of permanent magnets and the field induction in this chamber is 0 T.
Preparing the cell lysates
After 24 h, the cells were detached from the surface of the culture vessel using a trypsin/EDTA solution. After trypsin neutralization, the cells were centrifuged for 10 min at 2000 RPM, the supernatant was removed, and the cell pellets were washed with a PBS solution and used to prepare the cell lysates. The composition of the lysis buffer was a protease inhibitor (1.4 mg) and a phosphatase inhibitor (10 µL) that had been dissolved in 1 mL of PBS. The tubes containing the suspension of studied material in a lysis buffer were placed in liquid nitrogen for 30 min and stored at − 80 °C until further analysis. All of the studied biochemical parameters were recalculated for 106 cells.
Determining the superoxide dismutase activity (SOD)
SOD activity was determined using a Cayman Chemical's Superoxide Dismutase Assay Kit (Cayman Chemical, USA) and were performed according to the manufacturer's protocol. Previously prepared cell lysates were used as the test material, centrifuged after thawing and the collected supernatant was used for further studies.
The SOD activity was determined spectrophotometrically at 440–460 nm. Xanthine and hypoxanthine generate superoxide radicals which, when bound with a tetrazolium salt, transform it into red formazan. One unit of SOD activity is defined as the amount of the enzyme that is needed to convert 50% of the superoxide radicals.
Determining the glutathione peroxidase activity (GPx)
GPx activity was determined using a Cayman Chemical's Glutathione Peroxidase Assay Kit (Cayman Chemical, USA) and were performed according to the manufacturer's protocol.
The GPx activity was also evaluated based on the spectrophotometric method. The GPx activity was indirectly measured using a coupled reaction with glutathione reductase (GR (glutathione reductase). GPx catalyzes the reduction reaction of cumene hydroperoxide and as a result, an oxidized form of glutathione is formed, which was then reduced in the presence of GR and NADPH oxidation to NADP + (accompanied by a decrease in absorbance). The decrease in A340 absorbance is directly proportional to the GPx activity in the sample.
Determining the catalase activity (CAT)
The CAT activity was measured using a Catalase Assay Kit (Cayman Chemical, USA). The method is based on the reaction of CAT with methanol in the presence of an optimal concentration of H2O2. The formaldehyde that was produced was measured spectrophotometrically using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to purple.
Total RNA was extracted using a TRIzol reagent. The RNA extracts were treated with DNase I (RNeasy Mini Kit, Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quality of the extracts and the RNA concentration were determined as was previously reported .
Quantitative real-time RT-PCR assay
The gene expression of SOD1 (superoxide dismutase 1), SOD2 (superoxide dismutase 2), GPX1 (glutathione peroxidase 1), GSR (glutathione reductase), CAT (catalase) and β-actin were evaluated using real-time RT-qPCR and SYBR Green I chemistry (SYBR Green Quantitect RT-PCR Kit; QIAGEN, Valencia, CA, USA) as was previously described .
All of the samples were tested in triplicate. β-actin was also included as an endogenous positive control of the amplification and integrity of the extracts in order to monitor the RT-qPCR efficiency. Wells that did not contain a template were run as the negative controls. The thermal profile for the one-step RT-PCR was as follows: reverse transcription at 50 °C for 30 min, denaturation at 95 °C for 15 min and 40 cycles consisting of the following temperatures and time intervals: 94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s.
All data are expressed as the mean ± the standard deviation. An ANOVA and Tukey’s post hoc test were used to evaluate the results of the experiments. The statistical calculations were performed using STATISTICA 12.0 and the statistical significance was defined at p < 0.05.