A20 Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells to 60Co-γ Ionizing Radiation

Abstract

The radioresistance of hepatocellular carcinoma (HCC) cells is a critical obstacle for effectively applying radiotherapy (RT) in HCC treatment. NF-κB, an important transcription factor, can influence critical cell fate decisions by promoting cell survival or anti-apoptosis in response to cell-stress, e.g. chemotherapies or ionizing radiation (IR). A20, also named as tumor necrosis factor α induced protein 3 (TNFAIP3 ), is a dominant negative regulator of NF-κB pathway and its functions in HCC are largely unknown. Cell culture and adenovirus vectors L-02, HepG2, MHCC-97H (a highly aggressive HCC cell line) and MHCC-97L (a lowly aggressive HCC cell line) cells were purchased from cell resources center of Chinese Academy of Medical Sciences (Beijing, China). The adenovirus vectors containing full length sequence of A20 and its A20 siRNA was purchased from Vigene Company (Jinan, Shandong Province, China). The HCC specimens and the adjacent non-tumor tissues were obtained by routinely surgical operations and preserved by our lab. The collection of tissues and the study protocol was all approved by the Ethics Committee of Xi’an Jiaotong University School of Medicine, and with the informed consent of patients. Paired HCC and non-tumorous tissues (40 paired) were obtained from surgical resections from February 2015 to April 2016. Using such methods as qPCR; Western Blot analysis; 60Co-γ ionizing radiation and colony formation; TUNEL reaction; Apoptosis analysis; Transwell analysis; In vivo tumor growth. Higher expression of A20 was detected in hepatic non-tumor cell line or clinical specimens compared with HCC cell lines or clinical specimens. A20 decreased the expression of proteins mediating cellular stress/injury response or epithelial-mesenchymal transition (EMT) process. Overexpression of A20 via adenovirus enhanced the effect of 60Co-γ ionizing radiation (IR) on HCC cells’ injury, e.g. G2/M arrest or DNA double strands break (DSB). Moreover, A20 also enhanced the in vitro or in vivo survival inhibiting of HCC cells induced by IR. Our results showed A20 downregulates the protein level of cellular stress/injury response regulators and by then potentiate the susceptibility of HCC cell line to 60Co-γ IR. Overexpression of A20 via an adenoviral vector can be a useful strategy for increasing tumor radiosensitivity.