Study On The Radiosensitization To Breast Cancer Cells Induced By The Tat-SmacN7Fusion Peptide

Objective The purpose of this experiment was to investigate the radiosensitivity effect of Tat-SmacN7on human breast cancer cell line of SK-BR-3cells and its preliminary mechanism.Methods (1) The SK-BR-3cell line in the logarithmic phase was divided into4groups:the control group, Tat-SmacN7group, y-ray radiation group and combined group (y-ray radiation+Tat-SmacN7). MTT assay was.performed to evaluate the cytotoxicity. Colony formation assay was used to measure cell survival fraction. A single-hit multi-target model was used to fit cell survival curve and calculate the sensitive enhancement ratio(SER).(2) The apoptosis rate was analyzed using flow cytometry. The effect of DNA Double-strand Breaks caused by Tat-SmacN7combined with y-ray radiation was observed by commet assay.(3) Changes of XIAP, cIAP-1protein expressions and mRNA levels were detected by Western blot assay and RT-RCR.(4) Activity changes of caspase-3,8,9were observed by Western blot.Results (1) Tat-SmacN7inhibited cell grouth in a dose-dependent manner (r2=0.924, P<0.05)and its50%inhibition concentration (IC50) was (62.62±1.19) μmol/L, and IC20(5.66±0.67) μmol/L.The concentration5μmol/L could enhance cell radiosensitivity with a SER of1.48.(2)48hours post6Gy y-ray radiation,the incidence of apoptosis in the combined group was higher than that of irradiation alone group(P<0.05).6hours after irradiation,the tail moment of the combined group was significantly longer(P<0.01) than irradiation group.(3)Tat-SmacN7only inhibit XIAP expression, whereas Tat-SmacN7combined with radiation inhibit both XIAP and cIAP-1.(4) Compared with the irradiation group and Tat-SmacN7group, the combined group caspase-8, caspase-3, PARP activation were enhanced,whereas the expression and activation of caspase-9was no difference between the four groups.Conclusions Tat-SmacN7has a radiotherapy sensitization effect on human breast cancer SK-BR-3cell by promoting apoptosis and inhibiting DNA repair after irradiation.What’s more, the Pro-apoptotic effect was related with downregulating XIAP mRNA,promoting cIAP-1degradation and activating caspase pathway.