Study On Radiosensitization Of Tumor Cells Caused By Tat-SmacN7 Fusion Peptide

Tumor is one of diseases which seriously threaten human health. There are several methods for tumor therapy in clinic, including surgery, radiotherapy, chemotherapy, biotherapy, and some auxiliary methods, such as the traditional Chinese medicine treatment, endocrine therapy, radio frequency ablation therapy and thermotherapy, et al. Radiotherapy is more and more popular nowadays in these methods. The radiosensitization of tumor is different because the occurrence and the development of tumor is a complex process involving a multi-factor and multi-stage. Additionally, radioresistence may be developed by tumor cells to radiotherapy, which will result in reduction of therapy efficacy. Thus, it is very important to improve the sensitivity of tumor cells to radiation.Abnormal regulation of apoptosis signaling pathways is the main reason of tolerance in tumor cells to radiation therapy. The mechanism of radioresistance of IAPs in tumor cells is to suppress apoptosis by binding to caspase-3,-7 and-9, and inhibiting their activity. XIAP, cIAP-1 and cIAP-2 are important members in IAP family proteins. Smac/DIABLO, a protein released from mitochondria into cytokinesis in response to apoptotic triggering, can interfere the inhibiting activity of IAP to caspases, thus facilitating the apoptotic pathway and promoting the radiosensitization. SmacN7 is the active domain of Smac protein which has the apoptosis-promoting function. As SmacN7 can’t enter the tumor cells freely, we use the Tat-SmacN7 fusion peptide which can enter the cell to explore the mechanism of radiosensitization for tumor cells.Objective:To discover radiosensitization of Tat-SmacN7 to human esophageal cell line (EC 109) and non-small cell lung cancer (NSCLC) cell line NCI-H460 (H460). Additionally, the mechanism of radiosensitization was to be explored.Methods:(1) The cells were assigned to four groups as following:Control, Tat-SmacN7 (20μmol/L), Radiation (4 Gy) and Tat-SmacN7+Radiation. Clonogenic assay and flow cytometry for determination of apoptosis were used to examine radiosensitization of Tat-SmacN7 to the two cell lines. (2) PCR and Western blotting assay were used to examine the RNA levels of caspases and the protein expression of XIAP. Caspase activation on Tat-SmacN7 radiosensitization was detected by caspase activity assays based on ELISA. Caspase inhibitor was used to block Tat-SmacN7 radiosensitization. (3) Tumor growth was observed in nude mice in different treated groups in vivo experiments.Results:(1) Tat-SmacN7 was discovered to have radiosensitization in the two cell lines such as radioresistant (H460) and radiosensitive cells (Ec109). The sensitization enhancement ratios (SER) were 1.61 and 1.53 respectively at micromolar concentrations. However, it was not observed that Tat-SmacN7 could promote apoptosis if used as a single agent. (2) Significant increase was found in mRNA expression and activity of caspase-3,-8 and-9 in the combination treatment group based on PCR and ELASA experiments. The pan-caspase inhibitor-z-VAD-fmk significantly decreased apoptosis in both cell lines with SER reduction from 1.63 to 1.17 in H460 cells and 1.51 to 1.09 in Ec109 cells treated by Tat-SmacN7 and radiation. (3) As in vivo experiments, remarkable suppression of tumor growth was discorved in the combination treatment group of Tat-SmacN7 and radiation, which is statistically significantly greater than any treatment alone.Conclusions:It is reported here that Tat-SmacN7 is a novel radiosensitizer to tumor cells, and with low toxicity in cells. The mechanism of Tat-SmacN7 radiosensitization involved improvement in mRNA expression and activity of caspases. Our study provides the proof-of-concept for future development of Tat-SmacN7 as a new radiosensitizing agent against cancer cells.