The studies of free radicals in melanin and the human melanoma malignum cells by an X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy were presented. The original results were compared with those published earlier. The aim of this work was application of the advanced spectral analysis to determine free radical properties in melanin biopolymers obtained from different melanotic tumor cells and free radicals existing in the human melanoma cells. Magnetic spin-lattice interactions in melanin samples were tested. The evolution of lineshape of tumor cells with increasing of microwave power was determined to confirm their complex free radical system. The useful shape parameters were proposed. The shape of melanotic tumor cells was analyzed. EPR spectra of free radicals in the melanin isolated from different tumor cells measured in the wide range of microwave power were analyzed. The melanins were obtained from the control tumor cells and the cells cultured with the several antitumor substances. The usefulness of the electron paramagnetic resonance spectroscopy was confirmed.
- human melanoma malignum cells
- free radicals
- paramagnetic centers
- electron paramagnetic resonance spectroscopy
Free radicals of natural melanin and the melanin in the human
Eumelanin was found in the melanotic tumor cells [9, 10]. Melanotic tumor cells are studied by NMR [11, 12], FTIR [13, 14], and HPLC [15, 16] methods. In this work we were interested in EPR studies of melanin from different tumor cells. Melanin polymers are known from paramagnetic character and o-semiquinone free radicals with spin S = 1/2 [17–40]. Unpaired electrons of free radicals obey the electron paramagnetic resonance (EPR) effect [17–40]. o-Semiquinone free radicals absorb microwaves in the magnetic field. This absorption is the base of electron paramagnetic resonance (EPR) spectroscopy [41–43]. Free radicals in eumelanin [17–19, 26, 40] and pheomelanin [22, 31, 38–40] are responsible for the EPR spectra, which differ in the shape. Typical EPR spectra of the model eumelanin DOPA-melanin and pheomelanin are shown in Figure 2. Comparison of the lineshape of these EPR spectra indicates that eumelanin reveals the simple single line (Figure 2a) and EPR line of pheomelanin reveals the complex shape with the unresolved hyperfine structure (Figure 2b). The lineshapes of the EPR spectra of DOPA-melanin [17–19, 26, 34–37] and pheomelanin [22, 31, 38–40] were presented in a lot of papers. EPR spectra were measured for free radicals in melanotic tumor cells [44–52].
The drugs and substances with the antitumor interactions are still developed and searched [53, 54]. Melanin by free radicals interacts with drugs [55–57]. The effect of the antitumor substances on free radicals in the human tumor melanotic cells was shown by the use of the EPR spectroscopy [58–60].
The aim of this work was application of the advanced spectral analysis to determine free radical properties in melanin biopolymers obtained from the melanotic tumor cells and free radicals exiting in human melanoma cells. Free radicals in the original melanin samples and samples treated by the several antitumor substances were studied. The physical method of free radical detection based on paramagnetic character of melanins was used. EPR spectra of the tested natural melanins were compared with those of the model synthetic melanin polymers.
The innovatory lineshape analysis and the influence of microwave power on the complex EPR spectra were performed. The results were useful in medicinal therapy of the melanotic tumor cells. Both our published quantitative results [58–60] were cited, and the original spectral unpublished results were presented. The novelty in the present work, relative to our earlier papers [58–60], was the proposition of the spectral parameters to examine of the multicomponent EPR spectra as the sum of lines resulted from different types of free radicals existing in the melanotic A-2058 cells. The changes of these parameters with increasing of microwave power for the EPR spectra of the control cells and the cells cultured with valproic acid (VPA), 5,7-dimethoxycoumarin (DMC), and both valproic acid and 5,7-dimethoxycoumarin were presented.
2.1. The tested antitumor substances
The influence of the following substances on human
2.2. The tested human
melanoma malignum cells
The three types of the human malignant melanoma cell lines, A-2058, A-375, and G-361, were used in this study. The cells were also cultured with the antitumor substances: valproic acid (VPA), 5,7-dimethoxycoumarin (DMC), and both VPA and DMC. In our EPR studies, the measurements were performed for the same number of cells.
The A-2058, A-375, and G-361 cells were obtained from LGC Promochem (Łomianki, Poland). A-2058 cells and A-375 cells were grown in the Minimum Essential Medium Eagle (Sigma-Aldrich). G-361 cells were grown in McCoy’s medium (Sigma-Aldrich). These media were supplemented by the following components: 10% fetal bovine serum (FBS, PAA), 100 U/ml penicillin (Sigma-Aldrich), 100 μg/mL streptomycin (Sigma-Aldrich), and 10 mM HEPES (Sigma-Aldrich). The cells were incubated at temperature 37°C with the use of 5% CO2. The incubation details were described in [58, 60].
The human malignant melanoma cell lines were incubated with 1 mM VPA, 10 μM DMC, and their combination for 4 days (A-2058) or 7 days (A-375 and G-361). EPR spectra of free radicals in the A-2058 cells and in melanin isolated from A-375, and G-361 cells were analyzed.
2.3. Isolation of melanin biopolymers from the melanotic cells
Melanin was isolated from the human
2.4. The model eumelanin
The model eumelanin as DOPA-melanin was obtained by tyrosinase-catalyzed oxidation of 3,4-dihydroxyphenylalanine. The precursor (3,4-dihydroxyphenylalanine) was obtained from Sigma-Aldrich firm. The precursor was dissolved in 50 mM sodium phosphate buffer (pH 6.8). The final concentration was 2 mM. The reaction mixture after addition of tyrosinase (100 U/ml) was incubated for 48°C at temperature of 37°C. DOPA-melanin was obtained from the mixture by centrifugation (5000 × g, 15 min). The samples were washed by deionized water. Tyrosine was removed from melanin sample by treatment with SDS and methanol and NaCl. Finally, the sample was rewashed with deionized water and dried to a constant weight at temperature 37°C. This procedure was described in detail in [59, 60].
2.5. EPR measurements
2.5.1. EPR detection system
Free radicals in melanin biopolymers existing in different types of tumor cells and model synthetic melanin were examined by the use of electron paramagnetic resonance (EPR) spectroscopy. EPR spectra of melanin isolated from the cells and EPR spectra of the whole melanotic cells were tested. The first-derivative spectra were measured by an X-band (9.3 GHz) EPR spectrometer produced by Radiopan (Poznań, Poland) and the numerical data acquisition system—the Rapid Scan Unit of Jagmar (Kraków, Poland) (Figure 4).
The cells or melanin samples in thin-walled glass tubes were located in the resonance cavity in magnetic field produced by electromagnet of the EPR spectrometer (Figure 5). In the magnetic field, the Zeeman splitting appeared [41, 42]. Free radicals absorb microwaves according to the electron paramagnetic resonance condition [41, 42]:
The absorption is proportional to the free radical concentrations in the samples. The detailed determination of the free radical concentrations in cells and melanin samples was described in [58–60].
For the measurements and spectral analysis, the professional spectroscopic programs of Jagmar (Kraków, Poland), LabVIEW 8.5 of National Instruments (USA) and Origin (USA) were used. The Silesian Medical University has the right to use these programs. The program to spectroscopic analysis was prepared by Jagmar firm specially to our EPR spectrometer. The other programs are widely available.
2.5.2. The parameters of the EPR measurements
The EPR spectra were measured with the magnetic modulation of 100 kHz. Microwave frequency (
The maximal microwave power produced by klystron in microwave bridge of the EPR spectrometer was 70 mW. The measurements of the EPR spectra were done in the range of microwave power from 2.2 mW (attenuation of 15 dB) to 70 mW (attenuation of 0 dB). The microwave power was regulated by attenuation according to the formula [41, 42]:
2.5.3. Analysis of the EPR spectra
The influence of microwave power in the range of 2.2–70 mW on the lineshape parameters of the EPR spectra of the tested samples was determined. The model first-derivative EPR spectrum with the values, A1, A2, B1, and B2, was shown in Figure 6. The lineshape parameters were obtained as A1/A2, A1−A2, B1/B2, and B1−B2.
The evolution of the proposed lineshape parameter with increasing of microwave power gives information about complex free radical system in the biological samples.
The influence of microwave power on the integral intensities (
The changes of integral intensity (
3. Results and discussion
3.1. EPR spectra of free radicals in the human
melanoma malignum A-2058 cells
Free radicals with the strong EPR lines of g-factor near 2 were found in A-2058 human melanoma cells . The EPR spectra of the A-2058 cells recorded with the attenuation of microwave power of 7 dB were presented in Figure 7. The other spectra of these samples were presented in paper . The EPR spectra are the broad nonsymmetrical lines (Figure 7). The broadening of the EPR lines of A-2058 cells is caused by dipolar interactions between free radicals. In this study we concentrated on the spin-lattice interactions in A-20058 cells and on their complex system of free radicals.
The influence of the antitumor substances, VPA, DMC, and both VPA and DMC, on spin-lattice interactions in A-2058 human melanoma cells was determined. The influence of microwave power (
Integral intensities (
The other situation was observed for the human malignant melanoma cell line A-2058 cultured with both VPA and DMC. The integral intensity (
The lineshape of the EPR spectra of the control A-2058 cells, and the A-2058 cells cultured with VPA, DMC, and both VPA and DMC, changed with increasing of microwave power (
All the tested lineshape parameters (A1/A2, A1−A2, B1/B2, and B1−B2) for the control A-2058 cells and for the A-2058 cells cultured with the antitumor substances (VPA, DMC, and both VPA and DMC) were not constant, and their changes with microwave power were observed (Figures 9–12). The strongest changes of the parameters A1–A2 (Figure 10) and B1−B2 (Figure 12) were obtained. The changes of the spectral shape parameters with microwave power were not regular (Figures 9–12). These nonregular changes of the spectral shape parameters with microwave power confirmed the existence of several types of free radical in the tested A-2058 cells, both in the control cells and in the cells treated with the used antitumor substances.
We proposed these shape parameters, A1/A2, A1−A2, B1/B2, and B1−B2, for checking the multicomponent type of free radical in cells. They supported in the analysis of complex free radicals in the other paramagnetic samples, for example, for drugs [64, 65]. The EPR spectra of the cells were superposition of several lines resulted from the individual groups of free radicals. The microwave power differently influenced these EPR components, dependent on the type of free radicals. Amplitudes (A), linewidths (ΔBpp), and integral intensities (
Besides the shape analysis proposed in this work, the important qualitative results for free radicals in the human
3.2. EPR spectra of free radicals in melanin isolated from human
melanoma malignum A-375 cells
Free radicals were also found in melanin biopolymer isolated from the control A-375 cells and the A-375 cells cultured with VPA, DMC, and both VPA and DMC. For all the melanin samples, EPR spectra were measured. The exemplary EPR spectra of melanin isolated from A-375 cells cultured with VPA and DMC, recorded with microwave power attenuation of 7 dB, were shown in Figure 13. The other EPR spectra of melanin originated from A-375 cells were shown in .
The parameters of the EPR spectra of the melanin obtained from A-375 cells changed with microwave power. In Figure 14, the influence of microwave power on integral intensities (
The integral intensities (
o-Semiquinone free radicals mainly existed in the melanin samples from A-375 cells. The quantitative results were published in the earlier paper [59, 60]. Considerable decrease of free radical concentration in melanin after treatment A-375 cells by both VPA and DMC was observed . Free radical concentration in melanin isolated from A-375 cells cultured with DMC was lower than in melanin from the cells cultured with VPA . The changes of amplitudes (
3.3. EPR spectra of free radicals in melanin isolated from human
melanoma malignum G-361 cells
EPR lines of o-semiquinone free radicals were also measured for melanin isolated from G-361 human melanoma cells. The EPR spectra of melanin isolated from the control G-361 cells, and the G-361 cells treated with VPA, DMC, and both VPA and DMC, measured with microwave power attenuation of 7 dB, were shown in Figure 15. The other spectra of these melanin samples were presented in paper . The high level of the noise was visible in these spectra (Figure 15), so the lower contents of free radicals were found in melanin from G-361 cells than from A-375 cells (Figure 13).
The influence of the antitumor substances, VPA, DMC, and both VPA and DMC, on spin-lattice interactions in melanin obtained from G-361 human melanoma cells was not stated. The changes of integral intensities (
The quantitative results of EPR examination of melanin originated from G-361 cells were described in paper . It was obtained that after treating of G-361 cells with both VPA and DMC free radical concentration in melanin strongly decreased . Free radical concentration in melanin isolated from G-361 cells cultured with DMC was higher than in melanin from the cells cultured with VPA . The changes of amplitudes (
The existence of o-semiquinone free radicals in melanin from the human
The presented electron paramagnetic resonance examinations of free radicals in melanin and tumor cells were supported by Medical University of Silesia, Katowice, Poland (grant no. KNW-1-005/K/6/0).
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