School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China.
Gansu University of Chinese Medicine, Lanzhou 730000, China.
Acta Biochim Biophys Sin (Shanghai). 2017 Nov 1;49(11):989-998. doi: 10.1093/abbs/gmx096.
The heavy ion beam is considered to be the ideal source for radiotherapy. The p53 tumor suppressor gene senses DNA damage and transducts intracellular apoptosis signals. Previous reports showed that the heavy ion beam can trigger complex forms of damage to cellular DNA, leading to cell cycle arrest and apoptosis of HepG2 human liver cancer cells; however, the mechanisms remains unclear fully. In order to explore whether the intrinsic or extrinsic pathway participates this process, HepG2 cells were treated with 12C6+ HIB irradiation at doses of 0 (control), 1, 2, 4, and 6 Gy with various methods employed to understand relevant mechanisms, such as detection of apoptosis, cell cycle, and Fas expression by flow cytometry, analysis of apoptotic morphology by electron microscopy and laser scanning confocal microscopy, and screening differentially expressed genes relating to p53 signaling pathway by PCR-array assay following with any genes confirmed by western blot analysis. This study showed that 12C6+ heavy ion beam irradiation at a dose of 6 Gy leads to endogenous DNA double-strand damage, G2/M cell cycle arrest, and apoptosis of human HepG2 cells via synergistic effect of the extrinsic and intrinsic pathways. Differentially expressed genes in the p53 signaling pathway related to DNA damage repair, apoptosis, cycle regulation, metastasis, deterioration and radioresistance were also discovered. Consequently, the expressions of Fas, TP53BP2, TP53AIP1, and CASP9 were confirmed upregulated after 12C6+ HIB irradiation treatment. In conclusion, this study demonstrated the mechanisms of inhibition and apoptosis induced by 12C6+ heavy ion beam irradiation on HepG2 cancer cells is mediated by initiation of the biological function of p53 signaling pathway including extrinsic and intrinsic apoptosis pathway.
重离子束被认为是放射治疗的理想源。p53 肿瘤抑制基因感知 DNA 损伤并转导细胞内凋亡信号。先前的报告表明，重离子束可以引发细胞 DNA 的复杂损伤形式，导致 HepG2 人肝癌细胞的细胞周期停滞和凋亡；然而，其机制尚未完全清楚。为了探讨内在或外在途径是否参与这一过程，用不同方法处理 HepG2 细胞，用 12C6+ HIB 照射，剂量分别为 0（对照）、1、2、4 和 6 Gy，以了解相关机制，如用流式细胞术检测细胞凋亡、细胞周期和 Fas 表达，用电子显微镜和激光扫描共聚焦显微镜分析凋亡形态，用 PCR-array 试验筛选与 p53 信号通路相关的差异表达基因，然后用 Western blot 分析确认任何基因。本研究表明，12C6+重离子束照射剂量为 6 Gy 可通过内外途径的协同作用，导致人 HepG2 细胞内源性 DNA 双链损伤、G2/M 细胞周期停滞和凋亡。还发现与 DNA 损伤修复、凋亡、周期调节、转移、恶化和放射抗性相关的 p53 信号通路中差异表达的基因。因此，在 12C6+ HIB 照射处理后，确认 Fas、TP53BP2、TP53AIP1 和 CASP9 的表达上调。总之，本研究表明，12C6+重离子束照射对 HepG2 癌细胞的抑制和凋亡机制是通过启动包括外在和内在凋亡途径在内的 p53 信号通路的生物学功能介导的。
