| background and purpose: Tongue squamous cell carcinoma（TSCC）is the most common malignancy in the oral cavity.At present,the clinical treatment of early-stage TSCCs primarily focuses on surgical therapy,whereas,concurrent chemoradiation is preferred for advanced stages,biotargeted therapy is supplementary.However,these methods have some deficiencies,such as the comparatively large surgical trauma was likely to lead to more complications,the tumour cells be less sensitive to chemo/radiotherapy,biologic medications are relatively expensive and can elicit an immune response.Therefore,new approaches for treating TSCCs with high efficiency and safety are required.For superficial tumors,hyperthermia is a physical therapy,which has fewer side effects.But there are disadvantages in traditional hyperthermia techniques,such as poor temperature regulation,temperature non-uniformity and it doesn’t observe therapeutic efficacy in real time.Meanwhile,whether there are any differences in the tumor cells’ adaptation to patterns of hyperthermia and the molecular mechanisms of tumor response to hyperthermia remain to be fully elucidated and requires further study.Based on what was stated,the study prepared a novel,soft and highly stable hyperthermia patch（HTP）based on unidirectional silver nanofibers（Ag NFs）network,and we determined the effectiveness of the hyperthermia using HTP to inhibit the growth of Cal-27 cells grown in vivo as subcutaneous xenografts in nude mice.We further explored optimal hyperthermia interval time to inhibit Cal-27 cells proliferation and the role of Notch signaling pathway during hyperthermia.Research methods:1.HTP was prepared by electrostatic spinning method and magnetron sputtering method.The morphology of HTP was studied by SEM.The real-time resistance was collected by Keysight 2700 multimeter,and the transmission spectrum was recorded by Hitachi spectrophotometer.During the heating process,the DC power supplied power to the HTP,and the temperature was checked by infrared thermal imager and infrared photos and videos were taken.2.A tumor-bearing nude mouse model of human tongue squamous cell carcinoma Cal-27 was established.When the average tumor diameter reached 5mm,the mice were randomly divided into experimental and control groups（10mice/group）,the control group was not given hyperthermia treatment,and the hyperthermia group was treated with HTP（42℃/45 min）for 7 days.During the heating treatment,the variations of tumor volume,body weight,food intake and water intake of nude mice in both groups were monitored.After the experiment,the tumor weight was recorded,and the morphological changes of transplanted tumor cells were observed by H＆E staining and Transmission electron microscopy section,Western Blot and immunohistochemistry were used to preliminarily explore the molecular mechanism of HTP inhibiting the growth of human tongue squamous cell carcinoma.3.Cal-27 cells were placed into a 42℃ incubator for 45 min,and the second heat therapy was performed respectively at 6 h,12 h,24 h,48 h and 72 h later.Cell activity was tested by CCK-8 assay at 0,12 h and 24 h after the end of hyperthermia.After determining the optimal hyperthermia interval,7 hyperthermia treatments were performed,and the CCK-8 assay and flow cytometry were used to detect the effects of hyperthermia on Cal-27 cells multiplication and apoptosis.The m RNA and protein expression quantity of Notch1,Jagged1 and Hes1 were analyzed by q RT-PCR assay and Western Bolt assay.Research results:1.HTP was successfully prepared and proved to be transparent,skin-sticking and highly stable.2.After hyperthermia treatment using HTP of Cal-27 cells in vivo as subcutaneous xenografts in nude mice,results showed that tumor growth in the hyperthermia group was significantly inhibited compared with the control group（P<0.05）;HE staining and transmission electron microscopy showed significant apoptosis in tumor tissues of hyperthermia group.3.The consequences of vitro experiments showed that hyperthermia at 42℃ for45 min every 12 h consistently inhibited the proliferation of Cal-27 cells（P<0.05）;in comparison to the control group,hyperthermia of the experiment group significantly inhibited the proliferation and induced apoptosis of Cal-27 cells after 7 times of heat treatment（P<0.05）.4.Western Blot and immunohistochemical experiments on transplanted tumor tissues showed that the expressions of the target molecules of Notch signaling pathway---Notch1,Jagged1 and Hes1 proteins were decreased in the hyperthermia group（P<0.05）.q RT-PCR and Western Bolt results of Cal-27 cells after hyperthermia showed that the m RNA and protein expressions of Notch1,Jagged1 and Hes1 in the hyperthermia group were also significantly lower than those in the control group（P<0.05）.Research conclusion: Both HTP hyperthermia and high-temperature cell incubator can induce tumor cell apoptosis and inhibit the growth of human tongue squamous cell carcinoma by continuous 42℃ of high temperature.The mechanism may be that hyperthermia can induce the apoptosis of Cal-27 cells by inhibiting Notch signaling pathway.These results will provide a novel efficient and safe clinical application for hyperthermia treatment of TSCC and suggest that Notch signaling pathway may be a potential target for hyperthermia to inhibit TSCC. |
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