Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma （ESCC）.We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay.The times T1,when the number of living cells reached a plateau and T2,when the number of living cells reached a new plateau after the addition of insulin were found.T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin （MDC）.Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively.Apoptotic ratio and fluorescent intensity were analysed by flow cytometry （FCM） quantitatively.Western blotting analysis was used to estimate the protein expression levels of AKT,mTOR,PI3K,PTEN,autophage related indicator LC3-Ⅱ and autophage related protein Beclin1 changes that occurred in the course of treatment.Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin.Insulin can increase the apoptosis induced by cisplatin.Apoptotic ratio of T1 plateau cells （（32.6±4.3）％） is significantly less than T2 plateau （（47.5±5.6）％）.MDC fluorescent intensity at T1 plateau （104.9±13.2） was significantly higher than intensity at T2 plateau （82.6±10.3）.After cotreatment with insulin,the expression level of LC3-Ⅱ,Beclin1 and PTEN in T2 plateau cells were significantly downregulated,but AKT,mTOR and PI3K expressions significantly upregulated compared with T1 plateau.Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells,which may be attributed to the anticancer effects of cisplatin.

Experimental Computed Tomography-guided Vena Cava Puncture in Pigs for Percutaneous Brachytherapy of Middle Mediastinal Lymph Node Metastases

Background： Percutaneous brachytherapy is a valuable method for the treatment of lung cancer and mediastinal lymph nodes metastasis. However, in some of the metastatic lymph nodes in the middle mediastinum, the percutaneous approach cannot be used safely due to possible damage to surrounding anatomical structures. We established an animal model （group of 12 pigs） to assess the safety and feasibility of computed tomography （CT）-guided vena cava puncture. Methods： Under CT guidance, an 18G needle was used to puncture the anterior wall of the anterior vena cava （AVC） in 12 pigs. The 18G needle was chosen as it is similar in size to the needles employed for clinical application in brachytherapy. The incidence of complications and vital signs was monitored during the procedure. Thoracotomy was performed to remove AVC specimens, which were analyzed for histological evidence of vessel wall damage and repair. Results： Following postoperative enhanced CT, two animals were found to have a small pneumothorax （one being hemopneumothorax）. The intraoperative oxygen saturation of both animals was not significantly decreased and was maintained at 93-100%. No animals developed mediastinal hematoma. Preoperative, intraoperative, and postoperative changes in blood pressure, heart rate, hemoglobin, and blood oxygen saturation were not significant. Histological evaluation of AVC specimens showed that by 7 days following the procedure, the endothelial layer was smooth with notable scar repair in the muscularis layer. Conclusions： CT performed after the procedure and histological preparations confirmed the safety of the procedure. This indicates that percutaneous brachytherapy for metastatic middle mediastinal lymph nodes can be carried out via the superior vena cava.