During the past decades, given the daily exposure to electromagnetic fields widely existing in the environment, uncertainty has persisted concerning possibly harmful effects of them on human health; as a result, many studies have strived to delineate such effects of magnetic fields from various aspects, which surprisingly provided contradictory results. Some of them report that the effect of weak magnetic fields on living systems is transient, and as the intensity of the field starts to decrease the system returns to its original state(18, 19). With the increasing number of patients diagnosed with cancer and a lack of definitive clinical success by the existing modalities in many cases, finding a new approach is of the essence(20).
Our previous study showed that SMF was able to increase the cytotoxic effects of cisplatin on viability percentage and reduce the resistance of A2780-CP cells through the production of verruca-shaped structures at the surface of the cell membrane(6). There have been many experiments regarding the evaluation of the possible genotoxicity caused by SMF, most of which indicated that the magnetic field alone had no effect on genotoxicity(11). Moreover, it has been reported that low to medium intensities of SMFs do not have much effect on cells; however, in the presence of external factors, they can give rise to the generation of free radicals and/or metal ions, and consequently, their effects become interestingly tangible. The combined treatment of cells with SMF and compounds such as Fecl3 has exhibited higher levels of lipid peroxidation and oxidative damage to DNA, all of which point to the synergistic effects of SMF on free radicals(11, 21).
It has been found that the amount of iron in cancer cells is higher than that in normal ones. By altering intracellular iron homeostasis, magnetic field can increase free iron within the nucleus and cytoplasm. Increased levels of iron can also increase hydroxyl radicals through the Fenton reaction(22, 23). Furthermore, cisplatin produces reactive oxygen species such as superoxide anion and hydroxyl radicals. An increase in reactive oxygen species causes lipid peroxidation of the membrane and eventually causes the inability of the antioxidant defense mechanism against damage caused by free radicals, hence genotoxicity(24). Similar to the aforementioned findings from other papers and as shown in Figs. 1 and 2, no considerable genetic damage was observed in both the resistant and sensitive groups treated with SMF alone. However, there were significant differences in DNA damage from that of the control group among those which received the cisplatin only at different times. In addition, SMF in combination with cisplatin at different times managed to result in more damage in the nuclear DNA content of A2780 cells in comparison with either the field or cisplatin alone. Similarly, resistant cells suffered higher levels of genotoxicity following being treated with the aforementioned combination at 24, 48, and 96 h. In both sensitive and resistant cells, the amount of damage gradually increased over time in groups treated with the drug alone as well as the combination. As shown in Fig. 3, the comet tail DNA% has been compared between both sensitive and resistant cell lines at different times and groups. It was only the sensitive group treated with the combination of cisplatin + SMF that had higher comet tail DNA% compared to that of the resistant group, whereas the other groups showed no statistically clear distinction from one another.
The cell cycle can be arrested by the combination of chemotherapy drugs and magnetic field, and subsequently, cells will be prevented from undergoing mitosis; as a result, the cell population in the G2/M phase increases, whereas the cell population in the G0/G1 phase decreases(25, 26). ATM protein is a serine-threonine protein kinase, the expression levels of which increases due to damage caused by either the direct effect of drugs or free radicals generated by magnetic field. It also activates regulatory proteins in the cell cycle, including E2F1, Cdc25, and p53 among others(27, 28). As shown in Figs. 4 and 5, following treatment with either drug alone or the combination, the percentage of cells in the G1 phase was reduced, and on the contrary, the G2 phase had been increased. Both the drug-resistant group and the sensitive group faced inhibition of cell division due to treatment with the combination, which eventually resulted in a significant increase in the percentage of cells in the G2 phase.
Some reports have demonstrated that the magnetic field may lead to a significant reduction in the levels of calcium ions in Bone marrow-derived stem/stromal cells, which can possibly decrease cell proliferation and increase the rate of apoptosis(29). There have been several studies showing that the magnetic field may affect the homeostasis of iron atoms in some cells and increase intracellular iron in the cytoplasm and nucleus. Such an increase will give rise to higher levels of hydroxyl radicals and as a result apoptosis(27). Apoptosis was found to be the predominant type of cell death following treatment with cisplatin only, and the rate of cell death had a direct relationship with the duration of treatment. Additionally, treatment with SMF along with cisplatin caused a meaningful increase in the rate of cell death in both cell lines, although in the case of resistant cells, a large fraction of cell death occurred through necrosis unlike that of the sensitive ones, being mainly apoptosis.
The results from real-time PCR indicated changes in the expression levels of some of the studied genes as shown in Fig. 8. The expression levels of the P53 gene, a well-known marker involved in apoptosis, had increased in both resistant and sensitive cell lines following treatment with either cisplatin alone or the combination. The expression levels of this gene in the groups of both cell lines that received the combination reached their highest level, which was consistent with the previous findings and might be one of the reasons justifying them. The expression levels of the P21 gene were also affected by different treatments and interestingly had the highest expression levels in groups treated with the combination. In the case of Bcl2 gene as one of the important anti-apoptotic proteins, there were no substantial changes in expression levels among most of the treated groups; except for the resistant group that was treated with the combination for 96 h, none of the others had significant changes in the Bcl2 gene expression levels compared to that of the control group. The results may suggest that the rates of apoptosis or other types of cell death have been affected by the combination mostly through a mechanism of action, including genes other than Bcl2. Several cancer studies have reported that higher expression levels of CTR1 are correlated with higher intracellular concentrations of pt-based compounds such as cisplatin and better prognosis following chemotherapy(30, 31). The results from the analysis of the expression levels of the CTR1 gene showed the great effect of the combination on the expression levels of this protein. As shown in Fig. 8, treatment of cells with SMF or cisplatin alone had no significant effect on CTR1 expression levels. On the contrary, after the combination of cisplatin + SMF, the expression levels of this protein in both groups, especially the resistant group, significantly increased. Considering these results, the increase in the uptake of cisplatin by cells that were measured in our previous study can be interpreted as a crucial element for overcoming the resistance of the A2780cp cell line.