| Background:Malignant melanoma is a kind of cancer generating from melanocytes,90%of which arises in the skin and a few cases occur in other sites. Statistical data showed that the incidence and mortality of malignant melanoma are continuously increasing worldwide and its incidence is rising steadily at a rate of4%per year, which is sharper than any other cancers. Melanoma constitutes only approximately3%of all skin cancers, but is the most deadly, accounting for approximately75%of all skin cancer-related death. When identified early and fully excised, long-term survival can be achieved. However, the median survival is less than1year in metastatic disease. The5-year survival rate of malignant melanoma is universally good in developed countries, but only40%in developing countries. Compared to the European Union and United States, malignant melanoma is rare in China, but it is usually present at advanced disease at diagnosis.A variety of effective treatments (including surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, etc.) have been successfully applied to malignant melanoma. The indications, contraindications and side effects of these treatments limit patient accessibility to some extent. In addition, some melanoma cases respond poorly to the traditional therapies. No standard treatments can be identified for patients with locally advanced or metastatic diseases not considered for surgery. Therefore, available current therapies do not add any survival benefit to these patients. In this regard, how to improve the overall survival of melanoma and develop new effective treatments with low toxicity continues to be a long-term topic for clinical and basic researchers. Cancer electrotherapy is a novel physical cancer treatment utilizing the electrical technology. Recently, with the rapid development of biological study of electricity, a series of new electric fields based physical therapies hold great promise for cancer therapy. Different electric fields with different parameters have been widely used to cancer treatments, in which the most used was the electric field used as radiofrequency or microwave devices killing the cells via hyperthermia. Recently, there are gradually increasing reports on the electric fields as physical therapies for cancer utilizing the non-thermal properties, in which the representative method is intermediate-frequency alternating electric field (IF-AEF). Data have been provided that at very low frequencies (under1kHz), alternating electric field stimulates excitable tissues, so that it has been claimed to stimulate bone growth and accelerate fracture healing. However, as the frequency of the electric field increases above1MHz, the stimulatory effect diminishes and heating becomes dominant. This phenomenon serves as the basis for some commonly used medical treatment modalities including diathermy and radiofrequency tumor ablation. Alternating electric field of intermediate frequencies (100kHz to1MHz) was considered not to have any meaningful biological effects. But some new reports showed that IF-AEF can also result in a number of anti-tumor effects. The reason why in the past we failed to find its anti-tumor effects was due to the strong frequency dependence of such effects. Not all IF-AEF can exert impact on the proliferation of special tumor cells, but only the IF-AEF at specific frequency will significantly affects the proliferation. When it deviates from the optimum frequency, its biological effects significantly reduce. Tumor-treating field is a kind of special IF-AEF found and termed by some researches from Israel. This IF-AEF at very low-intensity (<2V/cm) and intermediate-frequency (100-300kHz) can inhibit cancerous cell growth in vitro by an anti-microtubule mechanism. As a new physical cancer treatment modality, IF-AEF is effective when applied to human and animal cancer if the frequency is accurately selected. Previous studies have found that inappropriate treatment (knife, laser and freezing) may induce rapid growth of melanoma, so few studies were reported on the treatment of melanoma utilizing electric field or other physical technologies.Invasion and metastases are the most significant biological features leading to poor prognosis and the major cause of mortality. Malignant melanoma is accustomed to burrow into surrounding tissue, and then results in distant metastases. Therefore, inhibiting invasion is an important strategy for the cancer treatment and the key to therapeutic efficacy. Recently, some studies have reported that some electric fields have effects on cancer invasion. However, to our knowledge, the effects of IF-AEF on cancer invasion remain unclear. The occurrence and development of cancer are associated with abnormal apoptosis, in which the deficient apoptosis play a more important role than the excessive proliferation of cells. Many studies show that the levels of spontaneous apoptosis of melanoma cells are lower than many other malignant tumors, and melanoma cells are not susceptible to apoptosis induced by chemotherapy. Hence, if the anti-tumor technology is effective at initiating the process of apoptosis, then it can effectively inhibit cancer growth. It is well known that tumor development relies on a functioning vascular network to provide nutrient and oxygen, and tumors are unable to develop without tumor angiogenesis. Tumors can only remain dormant or even degradation with the support of blood vessels around them, because it is not enough. Initiation of angiogenesis, however, allows rapid tumor growth and ongoing tumor progression. Therefore, vascular-targeted antiangiogenic therapy is becoming a hot topic in cancer therapy. Rather than targeting the tumor cells per se, antiangiogenic therapy is intended to target the tumor blood vessels and disrupt support networks, and therefore inhibits tumor growth and metastasis. The current study has shown that the effects of different electric fields on tumor angiogenesis are significantly different, some inhibiting angiogenesis, whereas others may induce angiogenesis. The effects of IF-AEF on tumor angiogenesis have not been reported previously.Because the biological effects and underlying mechanisms of the electric fields are complex and restricted by many factors, therefore, further studies were needed to explore the mechanisms of IF-AEF. In the present study, we observed the effects of IF-AEF on the tumor cells and tissues from different biological levels. The effects of IF-AEF on the cell proliferation and invasion inhibition of malignant melanoma cells cultured were initially determined in vitro. Subsequently, the effects of IF-AEF on melanoma in mouse B16F10melanoma model and the underlying mechanisms were explored. Thus, this study will further elucidate the molecular mechanisms of IF-AEF, likely find novel therapeutic targets for malignant melanoma.Part I Effects of IF-AEF on the Proliferation and Invasion of B16F10cellsObjective To investigate the effects of IF-AEF on proliferation and invasion of malignant melanoma cells cultured in vitro.Methods Malignant melanoma B16F10cells were used. There are two parts, including MTT assay and Transwell invasion assay, in this study:(1) MTT assay:B16F10cells cultured in vitro were divided into control and IF-AEF groups. For this study we developed a special apparatus producing IF-AEF at the frequency from10kHz to500kHz and Vpp (voltage of peak to peak) from0V to30V. The IF-AEF parameters (frequency, Vpp and exposure time) were analysed in order to determine whether any effects was frequency, electrical intensity, or time-dependent. The experiment was divided into three parts. In the first part, IF-AEF inhibitory efficacy vs. frequency was studied. The Vpp of IF-AEF was set at30V and exposure time was48h. The IF-AEF groups were further divided into six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). We try to identify the frequency dependent of IF-AEF inhibitory efficacy and select the optimum frequency for next studies. In the second part, the optimum frequency (100kHz) of IF-AEF used and exposure time was48h. the IF-AEF groups were further divided into three Vpp subgroups (10V,20V,30V), in which the time of IF-AEF exposure was set at48h. In the third part, the IF-AEF groups were further divided into two different time subgroups (24h,48h), in which the Vpp of IF-AEF (100kHz) was set at30V. After exposure, plated samples were harvested and the cells were incubated in supplemented DMEM for24h before MTT assay and then the inhibitive rates of the cells were calculated.(2) Transwell invasion assay:B16F10cells cultured in vitro were divided into two groups: control and IF-AEF group. The IF-AEF groups were further divided into two time subgroups (24h,48h). B16F10cells cultured in vitro were exposured to IF-AEF (100kHz,30V) or sham exposure. Transwell chamber in vitro invasion assay was applied to observe the changes of invasion capability after treatment. Results (1) After treated with IF-AEF (Vpp=30V) for48h, the proliferation ability of B16F10cells was arrested at six six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). The inhibitive rates of cell growth were16.43±2.17%,27.59±3.44%,13.30±2.25%,7.08±1.03%,3.23±0.35%, and2.69±0.33%respectively. There is significant difference between six frequency subgroups (P<0.05) and the maximal growth inhibition was found at100kHz. So the optimum frequency of IF-AEF on the inhibition of B16F10cells growth was100kHz. After treated with IF-AEF (100kHz) for48h, the proliferation ability of B16F10cells was arrested at three Vpp subgroups (10V,20V,30V). The inhibitive rates of cell growth at Vpp of10V,20V and30V were4.37±0.51%,12.43±1.46%and27.10±3.49%respectively. There is significant difference between three Vpp subgroups (P<0.05). After treated with IF-AEF (100kHz) at Vpp of30V, the proliferation ability of B16F10cells was inhibited at two time subgroups. The inhibitive rate of cell growth at48h group was27.1±3.49%, which was remarkably higher than that of16.4±1.80%at24h group (P<0.05).(2) The result of transwell chamber in vitro invasion assay showed that in the transwell cell culture chambers, invading B16F10cells were significantly lower than those in the control group after treated with IF-AEF. The invasion ability of B16F10cells was expressed as the percentage of invading B16F10cells in the transwell chamber with or without matrigel. The invasion rate was49.79±9.12%,37.97±7.31%and27.64±5.09%at negative control group,24h group and48h group respectively. There is significant difference between each other (P<0.05).Conclusion (1) IF-AEF has inhibitory effects on proliferation of B16F10cells and100kHz IF-AEF cause maximal inhibition of B16F10cells growth. The effects of IF-AEF are frequency, intensity and time dependent.(2) IF-AEF can remarkably decrease the invasion ability of B16F10cells cultured in vitro. Part II Effects of IF-AEF on Subcutaneous Tumor Model of B16F10Cells in C57/BL6Mice and Underlying MechanismsObjective To observe the effects of IF-AEF on subcutaneous tumor model of B16F10cells in C57/BL6mice and explore the underlying mechanisms.Methods C57/BL6mice model bearing B16F10melanoma were successfully established and then the tumor-bearing mice were randomly divided into the two groups of control (n=10) and IF-AEF treatment (n=10). From2days after inoculation, the IF-AEF group was stimulated by the IF-AEF (sinusoidal wave,100kHz,30V) twice a day,4hr each time. The treatment was continued for7days, consecutively. Animals in the control group underwent sham treatment under the same schedule. Tumors were measured every3days after treatment, i.e. on days1,4and7after treatment. After7days, two mice from each group were sacrificed, the tumors were excised and samples were collected and processed immeditately for histopathological and immunohistochemical examination. The level of apoptosis was detected and we investigated the involvement of Bcl-2and Bax using immunohistochemistry on sections from both IF-AEF and untreated tumors. The tumor microvessel density and VEGF of the tumors were also tested by immunohistochemical staining. The level of expression of VEGF mRNA in the tumor of IF-AEF group and control group was tested by reverse transcription-polymerase chain reaction. To observe the survival rate, the remaining mice were kept alive and tumor growth was monitored.Results (1) After7days of IF-AEF exposure, there was a significant decrease in mean tumor volume in mice treated with IF-AEF compared with the control group (26.33±9.82mm3vs.263.79±107.34mm3, P<0.05). The mean survival time of the tumor-bearing mice in IF-AEF group was significantly longer than in control-treated animals (29.10±3.70days vs.23.60±3.70days, P<0.05).(2) Note marked differences in the extent of tumor necrosis regions between IF-AEF group and control group in HE staining examination. Tunel analysis showed that Tumors treated with IF-AEF exhibited significantly greater numbers of apoptotic cells than control tumors (31.74±6.62%vs.4.51±1.34%, P<0.05). In mice treated with IF-AEF, the expression of Bcl-2significantly decreased compared with the control group (P<0.05), as well as an increase in the expression of Bax (P<0.05).(3) IF-AEF treatment signficantly reduced the number of CD34-positive cells compared with controls group (23.42±5.53vs.13.46±2.97, P<0.05). During the reduction of MVD, we determined a decrease in the expression of VEGF. The level of expression of the VEGF mRNA decreased significantly as determined by RT-PCR in mice treated with IF-AEF compared with control-treated animals.Conclusion (1) IF-AEF causes a significant reduction in tumor growth rate without any significant side effects and extends survival period of C57/BL6mice bearing B16F10melanoma.(2) IF-AEF therapy of subcutaneous tumors results in a quantitative increase of B16F10cells apoptosis. The results of expression profile of apoptosis-relate gene suggest that the result may be associated with the decrease of Bcl-2and increase of Bax.(3) The application of IF-AEF to the tumors leads to a reduction in tumor MVD. The mechanism of action of the fields is, at least in part, dependent on inhibiting the functions of VEGF.
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