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doi   10.1700/912.10039
Tumori 2011;97(3):386-392




An investigation into the combined effect of static magnetic fields and different anticancer drugs on K562 cell membranes
Ying Liu1, Hao Qi1,2, Run-guang Sun1, and Wen-fang Chen1
1Biophysics Laboratory, College of Physics and Information Technology, Shaanxi Normal University; 2College of Life Sciences, Shaanxi Normal University, Xiían, China

Correspondence to: Prof Hao Qi, College of Life Sciences, Shaanxi Normal University, 199 South Changían Road, Xi'an, Shaanxi, PR China 710062.
e-mail qihao1846@sina.com

Received May 17, 2010;
accepted February 2, 2011.

Key words: anticancer drug, static magnetic field (SMF), K562 cells.

abstract

Aims and background. Cell membranes were shown to be sensitive to and affected by static magnetic fields (SMF).
Methods. Cells were treated with four anticancer drugs followed by treatment with a combination of drugs and SMF. Individual cells were examined using atomic force microscopy (AFM). The drugs were taxol (alkaloid), doxorubicin (anthracycline), cisplatin (platinum compound) and cyclophosphamide (alkylating agent).
Results. Holes were observed in cells exposed to SMF but not in control groups. The number, size and shape of the holes were dependent on the drug type, SMF parameters and the duration of exposure.
Conclusions. The results suggest that the application of a SMF could alter membrane permeability, increasing the flow of the anticancer drugs. This may be one of the reasons why SMF can strengthen the effect of anticancer drugs. Observations were also made of the effect of using different anticancer drugs. For example, the effect of SMF combined with taxol or cyclophosphamide on the cells was additive while the effect of SMF combined with cisplatin or doxorubicin was synergistic. The target sites of cisplatin and doxorubicin are nucleic acids; continued research is required into this important area to ascertain the effect of SMF on nucleic acids.

Introduction
Many researchers have observed the effects of static magnetic fields (SMF) on tumor cells, particularly the inhibiting effects. They have used SMF as an entry point for investigating biological effects. In order to reduce the toxicity and resistance of single anticancer drugs, a variety of unified treatments were required. The synergy of magnetic fields and anticancer drugs was one of the methods. It provides a new strategy for the effective treatment of cancer.
Many studies have shown that magnetic fields can inhibit the growth of tumor cells1-3. At the same time, the effects of anticancer drugs on cancer cells could be strengthened by combining the drug with an SMF4-6. Observing the surface of cells is an important issue in biomagnetic research; all the external factors acting on cells must at first act through the cell surface. At present, the equipment used for observing the surface structure of cells includes optical microscopy, electron microscopy, confocal laser scanning microscopy, and immunochemistry7. Atomic force microscopy (AFM) has not been used widely in biomagnetic research. Teodori et al.8 used electron microscopy, optical microscopy and AFM to research the effect of SMFs on the size, shape, orientation, and membrane surface of human glioblastoma cells. Their AFM procedures demonstrated that exposure to SMF not only affects the size, shape, and orientation but also the membrane surfaces of these cells. However, the AFM magnification chosen did not provide good images of cell surfaces. The advantage of AFM observation of fine structures was not fully realized.
Material and methods
AFM instrument model and scanning technique
We used a Shimadzu SPM-9500J3 WET CNII type atomic force microscope with a Si3N4 AFM probe and 100-µm-long cantilever. Its elastic coefficient was 0.06N/m. The scanning mode was tapping.
Cell culture
K562 cells were cultured in RPMI 1640 medium supplemented with 10% calf serum (volume fraction) and a double antibody (penicillin + streptomycin; final density 200 U/mL). Cells were grown at 37 °C in saturated water vapor containing 5% CO2 until the logarithmic phase.
Drug preparation and storage
We mixed 50 mg/mL taxol (Sigma-Aldrich) with DMSO configured as storage medium and refrigerated the mixture at -20 °C. The final concentration was 10 ng/mL. Ten milligrams of doxorubicin (Zhejiang Hai-zun Liquid Co., Ltd.) was dissolved in 10 mL normal saline. The solution was filtered and sterilized, and stored at -20 °C. The final concentration was 25 ng/mL. Cisplatin (Qi Lu Pharmaceutical Co, Ltd.) at a concentration of 1 mg/mL in normal saline solution was filtered and sterilized, and stored at -20 °C. The final concentration was 10 µg/mL. Cyclophosphamide (Fluka) at a concentration of 40 mg/mL in saline solution was filtered and sterilized, and stored -20 °C. The final concentration was 0.4 mg/mL.
Magnetic field
Cells were placed in a 9-mT magnetic field produced by a solenoid. The volume of the uniform magnetic field was 12 × 5 × 5 cm. The variation in field strength in this region was less than 1%. The magnetic field was parallel to the base of the container in which the cells were cultured. The ambient temperature was controlled in the range of 36~37 °C.
Sample preparation
K562 cells were gently rinsed twice with PBS (pH 7.2, 0.1 M), centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were fixed using 1.5% glutaraldehyde for 15 minutes, then the supernatant was discarded. The rinsing was repeated, then the cells were centrifuged at 4000 rpm for 5 minutes. These procedures changed the density to 1 × 106 cells/mL. This suspension was dropped onto mica made to fit the atomic force microscope. After drying, observations were made using the tapping mode of the atomic force microscope, open to the atmosphere.
Experimental groups
The MTT method indicated that the synergistic action of the SMF and 10 ng/mL of taxol or 0.4 mg/mL of cyclophosphamide occurred after exposure for 24 hours, and the synergistic action of the magnetic field and 10 µg/mL of cisplatin or 25 ng/mL of doxorubicin occurred after exposure for 12 hours. The effects of the drugs and SMF were studied in four treatment groups for each drug.
Taxol (10 ng/mL): control group (conventional cultivation 24 hours); simple magnetic treatment for 24 hours; 10 ng/mL taxol treatment for 24 hours; 10 ng/mL taxol and magnetic field treatment for 24 hours.
Doxorubicin (25 ng/mL): control group (conventional cultivation 12 hours); simple magnetic treatment for 12 hours; 25 ng/mL doxorubicin treatment for 12 hours; 25 ng/mL doxorubicin and magnetic field treatment for 12 hours.
Cisplatin (10 μg/mL): control group (conventional cultivation 12 hours); magnetic treatment for 12 hours; 10 µg/mL cisplatin treatment for 12 hours; 10 µg/mL cisplatin and magnetic field treatment for 12 hours.
Cyclophosphamide (0.4 mg/mL): control group (conventional cultivation 24 hours); simple magnetic treatment for 24 hours; 0.4 mg/mL cyclophosphamide treatment for 24 hours; 0.4 mg/mL cyclophosphamide and magnetic field treatment for 24 hours.
Experimental results
Taxol (10 ng/mL)
The cell surface in the control group was relatively smooth (Figure 1A), whereas the cells treated by the magnetic field had changed. A pore-like surface structure had developed and protruding structures of various lengths (0.1~0.5μµm) were also observed (Figure 1B, arrow). Large apophyses (0.3~1.3 µm) appeared on the cells in a regular arrangement (Figure 1C, ▲). Large holes with a diameter of 0.47 µm and much larger apophyses (1.85 and 2.04 µm in diameter) could be observed at the same time (Figure 1D, arrow). The arrangement of apophyses was irregular (Figure 1D, ▲).









Doxorubicin (25 ng/mL)
 The cells showed changes similar to those after taxol and magnetic field exposure. The pore-like structure measured 0.23 µm (Figure 2B, arrow). After treatment, areas had developed showing a granular structure of uniform size (0.15 µm) (Figure 2C, arrow). Certain regions showed surface depressions. Irregular holes (0.02~0.2 µm) with some protruding structures were observed (Figure 2D, arrow), all larger than the granular structure mentioned above.
Cisplatin (10 µg/mL)
The results of large-scale scanning (5 × 5 µm) are shown in Figure 3A-D. As with the other treatments, cells of the control group remained smooth (Figure 3A and 3E). After magnetic field exposure for 12 hours variously sized holes had developed on the cell surfaces (Figure 3F, arrow). After cisplatin treatment for 12 hours, many different sizes and shapes of holes had developed (Figure 3G, arrow). Further, verruca-like raised areas were distributed around the holes (Figure 3G▼). After magnetic field and cisplatin treatment, there were holes of different shapes on the cells (Figure 3H, arrow). The biggest hole, which had a strip-like structure, is shown in Figure 3D. The width of the hole was 0.81 µm and the length 1.76 µm (Figure 3D). The results indicated that the combination of magnetic field and cisplatin produced considerable surface damage.
Cyclophosphamide (0.4 mg/mL)
The control group cells were smooth. After SMF treatment for 24 hours, some holes appeared on the cells (Figure 4A). In contrast to the results of the previous experiments, longer exposure to SMF produced greater damage (Figure 4B, Figure 3F, H). After cyclophosphamide treatment, variously sized round holes appeared on the cells (Figure 4C). The edges of the holes on cells treated with cyclophosphamide were not as rough as those on cells treated by SMF. After combined cyclophosphamide and SMF treatment, the diameter of the holes increased and the roughness of the edges was intermediate between the cyclophosphamide treatment group and the SMF treatment group (Figure 4D).
Discussion
Effect of magnetic fields on the surface of the cells
There were some holes on the tumor cells after exposure to SMF. The number, size and shape was dependent on the exposure time. The results indicate that the holes produced by the action of SMF were related to the synergistic action of SMF and the anticancer drug. If SMF could make holes in the cell surfaces, much more anticancer drug would flow into the cells. These results suggest that SMF influenced tumor cells by altering their membrane permeability and increasing the flow rate of anticancer drugs. This may be one of the reasons why SMF appears to strengthen the effect of the drugs. Researchers have predicted that the synergistic action of SMF was related to the effect of the magnetic field on tumor cell membranes 6,9. However, these predictions needed to be substantiated by experimental evidence.



Effect of magnetic fields and anticancer drugs on the cell membrane
The anticancer drugs had different effects on the cell membranes. Taxol caused the formation of large apophyses with a regular arrangement (Figure 1C). Doxorubicin led to a granular structure of uniform size (Figure 2C). Cisplatin produced round holes of different sizes with verruca-like structures around the holes (Figure 3C). Cyclophosphamide produced round holes with smooth edges (Figure 4C).
Our research indicated that the holes on the cells appeared after SMF exposure. The diameter and the number of the holes increased as the exposure time to the SMF gradually increased. Different anticancer drugs have different effects on the cell membrane. These effects resulted in different synergistic actions of the magnetic fields combined with the anticancer drugs.
Taxol produced large, regular apophysis structures and cave-like structures on the cell surface. After combination with SMF, the apophyses remained. At the same time, a large number of holes appeared on the cells, similar to the holes appearing on the cells exposed to the magnetic field, but with smooth edges. All of this evidence indicates that the effect of the combination of SMF and taxol was additive.
Doxorubicin and SMF treatment produced large holes whose diameter was greater than those in the SMF-only group. Surface roughening also became evident. Most apophyses occurred at the edges of the holes. These results indicate that the combination of SMF and doxorubicin produces a greater level of damage than other combinations. Collectively, the studied phenomena suggested that the combined action of SMF and doxorubicin was synergistic (Figure 3D).
AFM images indicated that the cisplatin-SMF combination inflicted considerable injury on the cell surface, although with varying appearance. The round holes or hollows produced by cisplatin were fewer and pinhole-sized, and there were some adjacent rounded verruca-shaped structures. When cisplatin was combined with SMF, much larger holes appeared. These results indicate that the combination of cisplatin and SMF can increase the damaging effect of cisplatin on the cells.
The surfaces of cells were smooth when treated by cyclophosphamide; small holes with smooth edges were evident. After combination with SMF, the number of very small holes diminished. The cell surface retained the smooth structure produced by drug treatment, but the size of the holes increased. Hole edges became rough as in the SMF treatment group. This phenomenon suggested that the combination of SMF and cyclophosphamide had an additive effect.
In summary, we chose the above anticancer drugs because they performed synergistic action when we measured the activity of cells using the MTT method. The AFM images indicated that the combination of SMF with taxol and cyclophosphamide had an additive effect, whereas the combination of SMF with cisplatin and doxorubicin was synergistic. It should be taken into account that the target sites of cisplatin and doxorubicin are nucleic acids.
There is sufficient evidence in these results to warrant continued research into the effects of the combination of magnetic fields and anticancer drugs.
Conclusion
The surface of K562 cells treated with anticancer drugs combined with SMF was examined. Although the MTT method indicated synergistic action, AFM showed different results. The changes in the cell membrane were the outcome of additive or synergistic effects, and different anticancer drugs influenced the cell membrane in different ways. The combination of taxol and SMF reinforced the cell damage. However, it was just the sum of the effects of the magnetic field and taxol, and there was no heavier cell damage when we analyzed the fine structure of the cell membrane. The combination of SMF and doxorubicin was synergistic. When combined with cisplatin, SMF enhanced the noxious action of the drug on the cells. SMF combined with cyclophosphamide had just an additive effect, like taxol and SMF.
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