Effect Of Intermediate Frequency Alternating Current On Human Tumor Cells And Experimental Study Of Breast Cancer In

Objective:To investigate the effects of alternating current at intermediate frequency(ACIF) on cancer cells, Human glioblastoma cells U87 and human breast cancer cells MDA-MB-231 were chosen for the subsequent studies. The possible mechanisms of ACIF on cancer cells and the effects of ACIF on the sensitivity of cancer cells to chemotherapeutic drug were studied. The in vivo safety and efficacy of ACIF on mice were also evaluated.Methods:1. Optimized parameters to ACIF treatment for different cancer cells.We chose eight kinds of cancer cells for the study, the single variable method was used for optimal parameters. Different parameters of ACIF was applied, including frequency and duration. After 3 consecutive days, the survival rates of cells were determined by CCK-8 assay. According to the results, we chose U87 cells and MDA-MB-231 cells to investigate the time-effect relationship of ACIF. The same as above, CCK-8 assay was used.2. The efficacy of ACIF on cancer cells and the possible mechanismsAccording to the results above, we chose glioblastoma cells U87 and human breast cancer cells MDA-MB-231 to investigate the possible efficacy and mechanisms. After ACIF, Giemsa staining was applied to observe the changes in cell morphology, Flow cytometry was used to detect the intracellular ROS levels. For the proliferation, we first tested the growth curves of these two kinds of cells, and then flow cytometry after fluorescent labeling were used to test the cell cycle and division algebra, they were labeled with PI and CFSE, respectively. At last, we investigated whether ACIF can change the uptake of chemotherapy drugs. The qualitative analysis of intracellular drugs was measured by Confocal Laser Scanning Microscope(CLSM), Flow cytometry was used to quantitative analysis of it.3. The in vivo experiment of ACIF on human breast cancer(MDA-MB-231)The tumor-bearing mice model was established by transplanting breast cancer cell lines MDA-MB-231 into the subcutaneous tissue of the left forelimb of BALB/c nude mouse. All animals were divided into three groups randomly:control group, chemotherapy group(epirubicin hydrochloride) and combined group(electrical stimulation combined with chemotherapy). The control group was intraperitoneal injection of physiological saline 0.2 mL. The chemotherapy group was intraperitoneal injection of epirubicin hydrochloride 0.2 mL,8 mg/kg. The combined group first injected drug as the chemotherapy group, and then applied for ACIF for 50 kHz,45 min, The electrical stimulation carried out once every other day, at a total of seven times. The mice weight and tumor volume was measured for plotted the corresponding curves. When the experiment ended, mice were sacrificed, we weighted the tumors and other organs, Histopathologic Examination (HE) was also performed.Results:1. The results of CCK-8 assay showed alternating current at intermediate frequency has different effects on different cells. ACIF had no inhibition to U87 cells, but can promote its growth slightly. ACIF had inhibitory effect on human breast cancer cells MCF-7 and MDA-MB-231, especially MDA-MB-231. The inhibition rate of it was about 25%, the parameters at that time were 20-50 kHz,150 mA.2. The results of Giemsa staining results showed that ACIF could change the cell morphology to a certain degree. After ACIF, the two kinds of cells turned out to be rounder, especially for the MDA-MB-231. Further more, the outline of cells turned fuzzy, vacuoles were observed intracellularly. At the same time, ACIF raised the Reactive Oxygen Species(ROS) in a certain degree, it means some oxidative damage of cells, but it’s not significant. The results of growth curves also showed that ACIF has no inhibition on U87 cells, but it had inhibition on MDA-MB-231 cells. The cell cycle showed no significant differences on two kinds of cells. Flow cytometry showed the apoptosis index is 3.87%±0.59% on control group,7.00%±0.37% on electric group of MDA-MB-231. What’s more, ACIF made the cell division algebra lagged with one generation. Analysis of qualitative (Confocal Laser Scanning Microscope) and quantitative (Flow cytometry) both showed that ACIF could promote the uptake of chemotherapy drugs of MDA-MB-231 cells. The same phenomenon was not found in U87 cells.3. The results of in vivo experiment showed that ACIF combined with chemotherapy drug can inhibit tumor growth in nude mice significantly, and it’s greater than chemotherapy drug alone. What’s more, the ACIF did not increase the toxicity, It means ACIF was safe and good.Conclusions:The alternating current at intermediate frequency (ACIF) can damage MDA-MB-231 cells and has inhibition on human breast cancer. The possible mechanisms are ACIF can change the morphology and structure of cells; it also lead to tiny oxidative damage, induce apoptosis; At the same time, ACIF could increase the intracellular drug concentration. However, ACIF has no significant efficacy on U87 cells. In addition, the results of in vivo experiment indicate that combining ACIF with chemotherapy drug may increase chemotherapeutic efficacy and sensitivity without increasing treatment related toxicity. Whether ACIF can be an effective adjuvant method and which kinds of cancer it can be applied still needs more research.