| ObjectiveThe aim of the present study was to develop a new, non-invasive cancer treatment method--Alternating Current at Intermediate Frequency (ACIF) treatment of human breast cancer. On the basis of optimistic parameters to the third generation of the equipment with independent research, the safety and efficacy in vivo and vitro were evaluated and the possible inhibition mechanism was investigated. Whether ACIF could increase the sensitivity of MCF-7to chemotherapeutic drugs was also researched. The results provided new experimental and theoretical basis for further study of safe and effective physical therapy techniques.Methods1. Safety assessment of ACIF treatment in vitroAccording to People’s Republic of China National Standard GB/T16886.5:regulations (Biological evaluation of medical devices-Part5in vitro cytotoxicity test), L929mouse fibroblast cells were chosen for the study. After applying ACIF treatment of different frequency30min per day,3consecutive days, the survival rates to cells were determined by CCK-8method.2. Optimization parameters to ACIF treatment for MCF-7In MCF-7human breast cancer cells for the study, based on a single variable method, different parameters of ACIF were applied, including frequency, current intensity, and treating time. After3consecutive days, the survival rates to cells were determined by CCK-8method. The result was just the optimal treatment parameters for follow-up experiments.3. Exploration to the possible mechanism of ACIF inhibition on MCF-7proliferationBy reading the relevant literature, a different research to explore the inhibition mechanism of ACIF on MCF-7was selected. Firstly, the effects of ACIF on MCF-7proliferation was further determined, including growth curve, cell cycle, division algebra. The method of CCK-8assay and flow cytometry after fluorescent labeling were used respectively. Cells in cell cycle were labeled with PI mark, and CFSE in division algebra.After the ACIF with optimum stimulation parameters, cells ambient pH and temperature was tested, as well as the intracellular ROS levels. Wright’s Giemsa complex staining was applied to observe the impact of MCF-7overall cell morphology. Scanning electron microscopy (SEM) was to observe the cell surface structure and transmission electron microscopy (TEM) was to observe the cell internal structure change. Lanthanum nitrate tracer method was to observe changes in MCF-7cell membrane permeability; flow cytometry after Rhol23marked change in MCF-7cell mitochondrial membrane potential.4. Effect of ACIF on the MCF-7sensitivity to chemotherapeutic drugsIn human breast cancer cells resistant mutants MCF-7/Adr for the study, the ACIF with optimal parameters was applied. The survival rates under different concentrations of chemotherapy drugs to cells were determined by CCK-8assay. IC50and drug resistant index were compared to verify whether ACIF could work drugs sensitizing effect.According to existing research, ACIF increased on MCF-7drug susceptibility mechanism was researched--impact on P-gp expression. Western Blot, flow cytometry and fluorescently confocal imaging after fluorescent labeling were used in experiment.5. Inhibition of ACIF on MCF-7in vivoMCF-7tumor cell was inoculated subcutaneously in the right hind of BABL/c nude mice to establish tumor-bearing mouse model. After tumor volume reached experimental requirements, mice were randomly divided into2groups:control group and experimental group. ACIF was applied in the experimental group for60min daily,28consecutive days. The weight and tumor volume of nude mice were recorded every other day. Tumor-bearing mice were dissected for histopathological analysis when the end of the experiment.Results1. ACIF had well-deserved biosecurityIn vitro, biological safety test results proved that ACIF does not affect the normal cell morphology and proliferation. In vivo, weight of the mice after treatment had no obvious difference from the control. Otherwise, ACIF had no pathologic influence on main organs of the mice in experimental group.2. ACIF could inhibit MCF-7proliferation in vitroCCK-8assay demonstrated that ACIF inhibited MCF-7proliferation. The highest inhibition rate was approximately20%with the treatment parameters of100kHz,50mA. However, in vivo, gross tumor volumes of the mice after treatment had no obvious difference from the control. The cause requires further study.3. Possible mechanism of ACIF inhibition on MCF-7proliferationACIF could inhibit MCF-7proliferation. The apoptosis rate after three consecutive days was about10%. Meanwhile, ACIF increased the number of cells in G0/G1phase and reduced the number of S-phase cells. Cell division algebra lagged with two generations. The results presented that ACIF not only induce apoptosis, but also affect the cell cycle phase distribution of cells.Different with DC therapy, ACIF couldn’t change cell survival environment pH and temperature and had no oxidative damage. Cell morphology reservation results showed that MCF-7cell volume was increased and the surface microvillus was curled or even disappeared. Vacuoles were observed intracellularly, especially significant swelling of mitochondria. Lanthanum nitrate and Rho123fluorescently labeled tracer method both displayed that MCF-7cell membrane permeability increases after electrical stimulation.4. ACIF could increase the effects of chemotherapy drugsACIF could reduce IC50resistance index of human breast cancer resistant strains (MCF-7/Adr) for doxorubicin. This result proved primarily that ACIF could increase the effects of chemotherapy drugs. Plus or synergistic effect was observed when doxorubicin combined with ACIF replied on MCF-7. It demonstrated ACIF significantly improve the therapeutic effect of chemotherapy. P-gp expression of resistance protein was decreased, but not statistically significantly. The speculated mechanism may be related to the change in cell membrane permeability.ConclusionsACIF could inhibit the proliferation of human breast cancer cells (MCF-7) safely and effectively. Its mechanism of action may be to change the structure and permeability of cell membranes, cell cycle, and induce apoptosis. While, ACIF does not change the pH value and temperature of cell survival environment, no oxidative damage was produced. Under the same treatment parameters, frequency alternating current has a certain sensitizing effect of chemotherapy, but the mechanism is not clear. ACIF technology provides a new way for the treatment of cancer research. |
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