| Cancer has become one of the three major killers which threatened to human health. Currently, chemotherapy is one of the primary means of tumor therapy. However, the side effects of antineoplastic drugs and multidrug resistant (MDR) of tumor cells often limit the effectiveness of chemotherapy. It has been reported that static magnetic fields (SMFs) not only inhibited the proliferation of cancer cells, but also enhanced the cytotoxity of anti-cancer drugs. This information suggested that the application of SMFs might be a supportive method for tumor chemotherapy, allowing for equivalent doses of anticancer drugs to produce a more curative effect and to help to reduce the dosage of drugs and associated side-effects. However, the synergistic effect of SMFs and anticancer drugs was few; the mechanism of the synergistic effect between SMFs and anticancer drugs remains unclear. Firstly, we investigated the cell viability of different cancer cells treated with SMF and SMF combined with chemotherapeutic drugs, then analyzed the change of cell cycle distribution, cellular DNA damage, cell surface morphology and inter ultrastructure of cancer cells, comparing the killing effects of SMF and SMF combined with chemotherapeutic drugs on several cancer cell lines. From these investigations, we try to provide an experimental evidence to illustrate the mechanism of SMF and SMF combined with anti-cancer drugs on cancer cells. We further investigated the mechanism of killing effects of SMF and cisplatin (DDP) on K562 cells by AFM, HPLC and flame atomic absorption spectrometry, providing a research basis for the application of SMF plus antineoplastic drugs.In this study, we analyzed and compared the biological effects of SMF on different cancer cell lines (human chronic leukemic cell line K562, human colon adenocarcinoma cell line SW480, human hepatocellular carcinoma cell line HepG2, human hepatoma cell line SMMC-7721 and murine hepatocellular carcinoma cell line Hepal-6) and primary cultured mouse hepatocytes through detecting the alteration of life processes and structures characteristics after cells were exposed or sham-exposed to SMF. On this basis, to understand the mechanisms of SMF and drugs on cancer cells, we analyzed the cell viability, cell cycle distribution, DNA damage, cell surface fine structure and inter ultrastructure of K562 cells and Hepal-6 cells after cells were treated with SMF with or without different anti-cancer drugs. We also observed the morphology of cellular DNA and detected the alteration of intracellular DDP concentration and the expression of P-glycoprotein (P-gp) after K562 cells were exposed to DDP in present or absent of SMF, the results showed that the mechanism of killing effects of SMF and DDP on K562 cells was correlated to the raise of intracellular DDP concentration. At last, we analyzed the cell viability, cell cycle distribution, DNA damage and cell surface fine structure of three kind of human cancer cells (SW480 cells, HepG2 cells and SMMC-7721 cells) after cells were treated with SMF with or without ADM and to gain an understanding on the possible mechanisms for the observed destruction. We obtained the conclusions as follows:1. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, single cell gel electrophoresis (SCGE)/comet assay, atomic force microscopy and transmit electric microscope were used to understand the biological effects of SMFs on different cancer cell line and primary cultured mouse hepatocytes and to gain an understanding on the possible mechanisms for the observed destruction. Experiments data showed that SMF inhibited the proliferation of cancer cells while the exposure time which caused SMF-induced the inhibition effects was different, for example, for K562 cells, the exposure time was 24 h; the time of both SW480 cells and Hepal-6 cells was 36 h; for SMMC-7721 cells, the time was 6 h; to HepG2 cells, it was 24h. We speculated on that this phenomenon was corrected to growth velocity of cancer cells. Rapid proliferation of cells was more susceptible to SMF exposure, such as K562 cells and SMMC-7721 cells. SMF have the cumulative effect on cancer cells. After SMF exposure, K562 cells was arrested at G2/M phase, Hepal-6 cells was blocked in S phase, the ratio of G1 phase was raised in HepG2 cells, but the distribution of cell cycle of SMMC-7721 cells and SW480 cells have few influence. These results suggested that effects of SMF on cell cycle distribution of cancer cells were related to the generation time of cell cycle. If the exposure time was shorter than the generation time of cell cycle, such as SW480 cells and SMMC-7721 cells, the change of cell cycle distribution of SMF-exposed cells was visible difficultly. Cellular DNA damage was detected by SCGE. The data showed that DNA damage was significantly increased after K562 cells were exposed to SMF for 24 h. For SW480 cells, HepG2 cells and Hepal-6 cells, the time of DNA damage initiated at 36 h. Cellular DNA of SMMC-7721 was damaged after cells were exposed for 9 h. SMF have no influence on attachment and spread of Hepal-6 cells. However, SMF blocked the attachment and spread of HepG2 cells. These data indicated that cell cycle of cancer cells was arrested by SMF, this effect involved in cellular DNA damage, as well as adhesion and microfilament cytoskeleton. The results of AFM observation showed that the modification of cell surface fine structure was earlier than the proliferation inhibition when cancer cells were exposed to SMF, for example, tiny holes appeared in the cell surface when K562 cells were exposed to SMF for 3 h, hole-like structures was visible after SW480 cells were exposed to SMF for 12 h. Surface roughness was increased in Hepal-6 cells which exposed to SMF for 24 h. Before the proliferation of cancer cells was inhibited by SMF, the cell surface fine structure have already modified, cell membrane is one of the sensitive cell organelles. When primary cultured mouse hepatocytes were exposed to SMF for 36 h, SMF has few influences on cell viability, cell surface fine structures and cell cycle distribution. These results suggested that the sensitivity of normal cells to SMF was lower than that of cancer cells.2. The cell viability, the distribution of cell cycle, DNA damage and cell surface fine structure were analyzed by MTT assay, flow cytometry, comet assay, and atomic force microscopy after K562 cells and Hepal-6 cells were treated with SMF combined with different chemotherapeutic drugs (cyclophosphamide (CP), Paclitaxel (PTX), adriamycin (ADM), cisplatin (DDP) and camptothecin (HCPT)). The experiments results showed that in the present of SMF, the cytotoxity of chemotherapeutic drugs on K562 cells were enhanced and the killing concentration of drugs was reduced. The efficient killing concentration of CP on K562 cells was reduced from 1.6 to 0.4 mg/mL. The efficient cytotoxic concentration of PTX was decreased from 50 to 10 ng/mL. For ADM, the efficient cytotoxic concentration was reduced from 35 to 25 ng/mL. The efficient killing concentration of DDP was reduced from 20 to 10μg/mL. However, for Hepal-6 cells, a combination treatment of SMF and antineoplastic drugs only enhanced the cytotoxic effect, but did not decreased the killing concentration of antitumor drugs. After K562 cells and Hepal-6 cells were treated with CP, PTX or ADM, cells were blocked at G2/M phase; cells treated with DDP were arrested S phase. SMF enhanced this effect which induced by these chemotherapeutic drugs. The results of comet assay showed that SMF enhanced cellular DNA damage which induced by anticancer drugs (CP, ADM and DDP, which target on DNA). The modification of cell surface fine structure was enhanced after cells were exposed to SMF and drugs.3. The morphology of cellular DNA, the change of intracellular DDP concentration and the expression of P-glycoprotein (P-gp) were investigated by AFM, high-performance liquid chromatography (HPLC), flame atomic absorption spectrometry and FCM in order to exploring the possible mechanism of killing effects of SMF plus DDP on K562 cells after cells were exposed to DDP in present or absent of SMF. The results of AFM observation showed that cellular DNA morphology in SMF+DDP group was differ from that in DDP group. Experimental observation showed that cellular DNA was thickened by SMFs and fragmented by cisplatin. Combined treatment of SMFs and DDP resulted in serious damages to cellular DNA, such as thinning, increased DNA breakage, massive crosslinking, and formation of mass-like structures. AFM results revealed that the DNA damage caused by SMFs or DDP was distinct. When K562 cells were treated with SMFs plus DDP, DNA damage was more intense. The results of HPLC and flame atomic absorption spectrometry showed that the platinum content of extracellular medium in SMF+DDP group was lower than that of DDP group (P<0.01). The P-gp expression induced by DDP was significantly increased compared with controls (P<0.05). In contrast, in the SMF+DDP group, the P-gp expression was decreased compared with the DDP group (P<0.05). The data indicated that DDP induced the expression of P-gp in K562 cells, but SMF reversed partly the P-gp expression induced by DDP, promoting the intracellular DDP accumulation. Taken together, our results showed that the 8.8mT SMF enhanced the cytotoxity potency of DDP on K562 cells, and the decrease in P-gp expression may be one reason underlying this effect.4. The biological effects and the possible mechanism of SMF plus ADM on SW480, HepG2 and SMMC-7721 were investigated by MTT assay, FCM, SCGE and ROS assay. The results of MTT assay revealed that a combination treatment of SMF and ADM not only could enhance the cytotoxity of ADM on cancer cells, but also reduced the killing concentration of ADM. The data of FCM showed that K562 cells and SMMC-7721 cells were arrested at G2/M phase; HepG2 cells and SW480 cells were blocked in G1 phase after cells were exposed to SMF combined with ADM. We speculated on that this phenomenon was correlated to the characteristic of cell proliferation. The results of SCGE and ROS assay revealed that cellular DNA damage is related to the increase of intracellular ROS level. Furthermore, the experiment data also revealed that the sensitivity of cancer cells to change of ROS level which induced by exogenous factors was depended on intracellular ROS level of cancer cells themselves. These data indicated that ADM and SMF promoted the generation of intracellular ROS, increasing the cellular DNA damage.Altogether, we verified that SMF have the inhibition effects on proliferation of cancer cells, and enhanced the cytotoxity of anticancer drugs on cancer cells. The mechanism may be as follows:1. SMF exposure induced the cellular DNA damage, influenced on the DNA replication. Especially, when SMF combined with chemotherapeutic drugs which target on DNA, SMF enhanced DNA damage which induced by these drugs. After cellular DNA damage was accumulated to a certain extent, there would be represent the inhibition of SMF on the proliferation or growth of cancer cells.2. SMF affected charged biological macromolecules. SMF maybe affects the ion movement in the cancer cells and the biological macromolecules or organelles whose charge distribution is asymmetrical, such as cell membrane, Ca+, and depolymerization or assembly of microtubules and microfilaments. When these molecules or ions moved in SMF, they will be affected by the Lorentz force, interfering ions motion and producing some biological effects. However, SMF have few influences on normal cells, for example, primary cultured mouse hepatocytes. We presumed that the effects of SMF on charged particles in cancer cells influenced the biological rhythm of cancer cells. After the accumulation, the influences will be representing a series of biological effects.3. SMF affects some of the protein which located on the cell membrane, such as P-gp.4. SMF exposure results in elevating the intracellular ROS level. The sensitivity of cancer cells to change of ROS level which induced by exogenous factors was depended on intracellular ROS level of cancer cells themselves. When the intracellular ROS level of cancer cells themselves is higher, the cancer cells will be more sensitive to the anticancer drugs which induced ROS production. After a treatment of SMF plus drugs, SMF can greatly reduce the concentration of anticancer drugs, which will help reduce the side effect of drugs on the body.
|
PDF Full Text Request