| Objective:The objective of the present study is to explore the inhibitory efficacy and underlying mechanisms of alternating micro-current at intermediate frequency(ACIF) and ACIF combined with chemotherapy to proliferation of human breast cancer cells (wild type or drug resistance) in vitro and in vivo. Besides, investigating the potential inhibitory efficacy of ACIF to rabbit breast cancer.Methods:In vitro experiment:The human breast cancer cells (MCF-7) were exposed to the ACIF of different frequency and different time. Test the MCF-7cell viability by the MTT assay and Cell Counting Kit-8assay (CCK-8assay); Test the cell cycles by the flow cytometry; In addition, observe the cell morphous by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).To investigate the inhibitory efficacy of ACIF combined with chemotherapeutic agent to MCF-7by CCK-8assay. Applying CCK-8assay to test the IC50of chemotherapeutic agent (Doxorubicin. HC1) to MCF/Adr, then calculate the resistance index.In vivo experiment:Establish the model of tumor-bearing female BALB/c nude mice, observe the inhibitory efficacy of ACIF alone and in combination with chemotherapeutic agent to tumor. Measure tumor volume and plot tumor volume curves every other day. After1month’ s observation, kill all the mice and perform safety and efficacy analysis. In addition, establish the model of tumor-bearing female rabbit, observe the inhibitory efficacy of ACIF to tumor. Measure tumor volume and plot tumor volume curves every other day. After1month’s observation, kill all the rabbits and perform safety and efficacy analysis.Results:In vitro experiment:CCK-8assay showed that administering ACIF of300kHz to MCF-7cells30min per day and3days continuously, the MCF-7cells were inhibited strongly, and the inhibition rate is high up to32.3%, the inhibition rate of100kHz under the same condition is40.6%, the inhibition rate of variable frequency reached51.1%, and there is statistically significant difference. MTT assay showed that administering ACIF of300kHz to MCF-7cells90min per day and3days continuously, the MCF-7cells were inhibited strongly, and the inhibition rate is high up to37.2%. What’s more, under the same condition, the longer time administering ACIF of300kHz to MCF-7cells every day, the higher concentration of lactate dehydrogenase were tested. The FCM results show that the proliferation index (PI) of ACIF group (47.38%) is lower than that of control group (53.82%), and the cells in G0/G1phase are more than that in the control group, the cells in G2/M and S phase are less than that in the control group. The SEM images show that after applying with ACIF, there were small holes in the cell membrane, which are about6μm in diameter. TEM images showed that there were few organelles and integral microvilli at the cell surface in control group; while after applying ACIF, the microvilli disappeared, and the cell plasma and organelles increased. CCK-8assay showed that the inhibitory efficacy of ACIF adjuvant chemotherapy is no stronger than ACIF alone to MCF-7cells, while, the dose of adjuvant chemotherapy is so low. The results of CCK-8assay show that the IC50of Doxorubicin. HC1to MCF/Adr is53.30μg/mL, and the IC50of Doxorubicin. HC1to MCF-7is0.875μg/mL, and the resistance index of MCF/Adr is61.In vivo experiment:The tumor-bearing female BALB/c nude mice model:compared with the control mice, ACIF can effectively inhibit tumor growth and without any significant side effect. ACIF in combination with chemotherapy increased the sensitivity and didn’t increase the toxic effect of chemotherapy. These all indicate that ACIF may be a safe and effective treatment. In addition, the tumor-bearing female rabbit model:there are no statistically significant differences between the tumors of control group and ACIF-treating group, though ACIF didn’t inhibit tumor growth, there is no any device-related significant side effect. It also indicates that ACIF may be a safe and effective treatment.Conclusions:ACIF can inhibit the proliferation of MCF-7cells effectively both in vitro and in vivo, accordingly inhibits human breast cancer growth. The mechanisms may be it changes the inner and outer morphous and subsequently influent the normal functions. In addition, ACIF alters the cell cycles, thus inhibit the cell proliferation. However, ACIF doesn’t increase the sensitivity and curative effort of chemotherapy significantly under the same condition. On the other hand, it is necessary to further study the underlying mechanism, and more experiments in vivo and clinical trials are needed. |
