As shown in Fig. 1a, we design a strategy to investigate the effective inhibitory in SMF with low magnetic intensity, on which the cells and mice are placed on the N-pole. Specially, the cells including A2780 and Skov-3 cells are incubated in a cell culture flask with 1 mm thickness to detect their factors. After the cells are injected into mice subcutaneously, the experimental tumor bearing mice are established. To ensure that the distance between the tumor and magnet surface is less than 1 mm, these mice are fixed on the surface of magnet constantly. Furthermore, a precise surface magnetic intensity is measured. As shown in Fig. 1b, three magnets with surface magnetic intensities of 0.2T, 0.4T and 0.6T are used to evaluate the relationship of their surface magnetic intensity and the distance. Figure 1c shows that the curve shape is approximate inverse function, whose intensity is decreased gradually with the increase of distance away from the magnet surface and to near zero at the distance of 10 cm no matter their initial magnetic field strength. Closer observation finds the surface magnetic intensity falls to 0.185T, 0.392T and 0.587T at the distance of 1 mm for 0.2T, 0.4T and 0.6T magnet, respectively. Based on the result, the controlled distance precisely for in intro and in vivo model is important, leading to the same actual experimental conditions.
A2780 cells and Skov-3 cells are used to establish an in vitro model of ovarian cancer cells in gradient SMF. Figure 2 is the confocal microscope images of the both ovarian cancer lines under gradient SMF about 0.2T, 0.4T and 0.6T. As shown in Fig. 2, there is a big different nucleus number in the same size within the vision field in different magnetic fields. Specially, as seen from these images, the number of two kinds of ovarian cancer cell nucleus is decreased with the increasing of SMF magnetic intensity, which indicates that both ovarian cancer cells are inhibited under SMF magnetic field. Additionally, there is an abrupt inhibition under 0.6T SMF compared to that of 0.2T and 0.4T. The reason could contribute to the appearance of compensatory hyperplasia and the increase of nucleus, which leads to the aging of apoptotic cells. The results demonstrate that SMF has a very good inhibitory effect on ovarian cancer cells, revealing that SMF is a great potential treatment of ovarian cancer.
Furthermore, CCK-8 is performed to evaluate the survival rate of two kinds of ovarian cancer cells under different SMF. As shown in Fig. 3, the magnetic intensity with 0.2T, 0.4T and 0.6T can reduce the survival rate of two kinds of ovarian cancer cells. Among them, the survival rate of A2780 cells Skov-3 cells for 24 hours is decreased by 2.3%, 4.7% and 13.5%, and 2.1%, 4.68% and 10.1%, respectively, for 0.2T, 0.4T and 0.6T SMF. In other words, the inhibition degree of 0.6T SMF is about 2–4 times that of 0.2T and 0.4T SMF, which shows the largest inhibition on the survival rate of A2780 about 13.5%. Compared with Fig. 2, the results mean that high magnetic field strength is sensitive to the inhibitory effect on ovarian cancer cell lines.
To better quantify the inhibitory effect on both ovarian cancer cell lines, we perform the apoptotic rate detection experiments on two ovarian cancer lines under the same conditions. Flow cytometry analysis about two kinds of ovarian cancer cells under different SMF reveal the effect of ovarian cancer cells apoptosis on 0, 0.2T, 0.4T and 0.6T magnetic intensity for 24 hours. As shown in Fig. 4, Q1 is mechanical damaged cell, Q2 is late apoptotic or necrotic cell, Q3 is living cell, and Q4 is early apoptotic cell. Additionally, A2780 cell early apoptosis rates are 7.1%, 10.4%, 10.3% and 9.1%, late apoptosis rates are 5.1%, 11.8%, 16.5% and 25.3%, and the total apoptosis rates are 12.2%, 22.2%, 26.8% and 34.4% under control, 0.2T 0.4T and 0.6T SMF, respectively. Moreover, the early apoptosis rates of Skov-3 cell are 5.8%, 14.2%, 16.5% and 18.6%, the late apoptosis rates are 6.0%, 12.4%, 21.0% and 24.8%, and the total apoptosis rates are 11.8%, 26.6%, 37.5% and 46.1% under control, 0.2T 0.4T and 0.6T SMF, respectively. Therefore, the results prove that the effect of gradient SMF on the apoptosis of ovarian cancer cells in different lines is different, and the apoptosis rate gradually increases with the increasing SMF intensity, showing a magnetic intensity-dependent relationship of ovarian cancer.
It is generally believed that the free radical content of ROS in cells determines the oxidative senescence and death of cells, which is the key factor to promote the process of apoptosis43–44. So, we explored the effect of SMF on the total active oxygen content of two ovarian cancer cells within 24 hours. Figure 5a is the result of ROS laser confocal microscopy images with the same inoculation concentration for two ovarian cancer lines. The DCFH-DA fluorescence intensity in a single cell is increased although the number of cells in the same area is decreased because of the external SMF according to Fig. 2. However, the quantitative analysis of the total ROS in the same concentration cells by flow cytometry is conducted, as shown in Fig. 5b. Compared with the control groups, the total ROS in A2780 cells is increased by 24.5%, 69.7% and 135.0%, respectively, and the total ROS in Skov-3 cells is increased by 17.8%, 62.5% and 112.7%, respectively. The result suggests that SMF can stimulate the production of a large number of ROS in ovarian cancer cells to promote apoptosis. The reason may be that ovarian cancer cells causes its own oxidative damage to oxidative stress of SMF, or ROS oxidizes intracellular macromolecules, destroys the normal function of macromolecules, and thus causes damage to cells and even death45–46.
Ca2+ plays an indispensable role in the signaling cascade reaction between cells, and it has been proved that increased intracellular Ca2+ level can accelerate the apoptosis process of tumor cells41–42. Therefore, the influence of intracellular Ca2+ level on two kinds of ovarian cancer lines under SMF of 0, 0.2T, 0.4T, and 0.6T for 24 hours. As shown in Fig. 6a, the laser confocal microscope shows that the number of cells in the same area is decreased and the Fluo-3 fluorescence intensity in a single cell gradually is strengthened, with the increase of SMF magnetic intensity, in both two kinds of ovarian cancer cells, Furthermore, the total intracellular Ca2+ is quantitatively analyzed by flow cytometry in Fig. 6b. Compared with the control group, the intracellular Ca2+ level of A2780 under 0.2T, 0.4T, and 0.6T SMF is increased by 8.7%, 27.5% and 68.8%, respectively, and that of Skov-3 increases by 12.6%, 37.5% and 82.7%, respectively. The result suggests that gradient homeostasis magnetic field can stimulate Ca2+ influx in ovarian cancer cells or change signaling factors between ovarian cancer cells to promote apoptosis of ovarian cancer cells47. Thus, the intracellular Ca2+ level of ovarian cancer is a magnetic intensity-dependent.
As we well known, several references have reported that cancer cells may evade apoptosis through decreasing calcium influx into the cytoplasm47. The reasons can be mainly shown as following: on the hand, it can be achieved either by the downregulation of plasma membrane Ca2+-permeable ion channels or by the reduction of the signaling pathways to activate Ca2+-permeable ion channels48,49. On the other hand, the defensive mechanism against apoptosis would involve the cancer cell adaptation for the reducing basal [Ca2+]-endoplasmic reticulum (ER) without the induction of pro-apoptotic ER stress response that usually accompanies ER luminal calcium imbalance50. Therefore, in the present experiment, based on the inhibition of SMF on two kinds of ovarian cancer cells and the detection of apoptosis-related factors (ROS and Ca2+) in cancer cells, we suggests that the intracellular Ca2+ concentration is increased stimulated by SMF, which activates the Ca2+-dependent nuclear factors of activated T cells (NFAT) protein through ER luminal calcium imbalance. The results could activate Ca2+-permeable ion channels. Furthermore, the activated Ca2+-dependent NFAT protein can stimulate the mitochondria production of a large number of ROS in ovarian cancer cells that oxidize the key cysteine residues of transient receptor potential A1 (TRPA1) to activate proteins to promote apoptosis. Therefore, as shown in Fig. 7, ROS can synergize with Ca2+ to promote apoptosis in cancer cell51.
Furthermore, the results of in vivo nude mice experiments on A2780 ovarian cancer further confirm the above results. As shown in Fig. 8, different results in different treatment groups indicate that the low SMF magnetic intensity can effectively inhibit the growth and proliferation of ovarian cancer cells. It is obvious that the size of the tumor in the control group is increased significantly during the experiment. Compared with the control group, three SMF treatment groups show significant inhibitory effect on ovarian cancer of A2780 line. Among them, 0.6T SMF treatment group has the most obvious effect. As shown in Fig. 8b, the mean tumor volume of 0.6T SMF group increases most slowly among the four groups, which shows that 0.6T SMF have considerable growth inhibitory effect on tumors. Furthermore, on the 31 day, mice are sacrificed and tumors are excised for weight. The tumor photograph and their volume and weight in each group after the treatment are shown in Figs. 8a, 8b and 8c. 0.6T SMF group also shows a higher inhibition activity than that of control, 0.2T and 0.4T SMF groups. The mean tumor weight in 0.6T SMF group (0.186 ± 0.073g) is smaller than that of control group (1.170 ± 0.109g, P < 0.05), 0.2T SMF group (0.689 ± 0.098g, P < 0.05) and 0.4T SMF (0.278 ± 0.018g, P < 0.05). The results indicate that the treatment with SMF displays a significant antitumor performance. Thus, the above results reveal that SMF are a powerful candidate for combining therapy of cancer in vivo.