Wang Yali, Sun Chao, Mao Aihong, Zhang Xin, Zhou Xin, Wang Zhenhua, Zhang Hong
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, People'sRepublic of China; Key Laboratory of Heavy Ion Radiation Medicine of Chinese Academy of Sciences, Lanzhou 730000, People's Republic of China; Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, People's Republic of China; School of Pharmacy, Lanzhou University, Lanzhou 730000, People's Republic of China.
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, People'sRepublic of China; Key Laboratory of Heavy Ion Radiation Medicine of Chinese Academy of Sciences, Lanzhou 730000, People's Republic of China; Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, People's Republic of China.
Life Sci. 2015 Feb 15;123:43-50. doi: 10.1016/j.lfs.2014.12.027. Epub 2015 Jan 13.
Previous studies in malignant cells have shown that irradiation-induced upregulation of telomerase activity, not only protected damaged telomeres, but also contributed to DNA damage repair by chromosomal healing and increased resistance to irradiation. The purpose of the present study was to investigate the radiosensitizing effect of telomerase inhibitor MST-312 and the corresponding mechanism in the human hepatoma cell line HepG2.
Cell proliferation, telomerase activity, cell cycle distribution, DNA damage and repair, expression of p53, mitochondrial membrane potential, and cell apoptosis were measured with the MTT assay, real-time fluorescent quantitative PCR, flow cytometry, immunofluorescence, western blots, JC-1 staining, and Hoechst 33258 staining, respectively.
MST-312 effectively inhibited telomerase activity and showed relative weak toxicity to HepG2 cells at 4 μM. Compared with irradiation alone, 4 μM MST-312 pretreatment, followed by X-ray treatment, significantly reduced clonogenic potential. Aggravated DNA damage and increased sub-G1 cell fractions were observed. Further investigation found that homologous recombination (HR) repair protein Rad51 foci nuclear formation was blocked, and expression of p53 was elevated. These led to the collapse of mitochondrial membrane potential, and enhanced the apoptotic rate.
These data demonstrated that disturbances of telomerase function could enhance the radiosensitivity of HepG2 cells to X-ray irradiation by impairing HR repair processes. In addition, telomerase inhibitor MST-312 may be useful as an adjuvant treatment in combination with irradiation.
以往对恶性细胞的研究表明，辐射诱导的端粒酶活性上调不仅能保护受损的端粒，还能通过染色体修复促进DNA损伤修复，并增强对辐射的抗性。本研究的目的是探讨端粒酶抑制剂MST - 312对人肝癌细胞系HepG2的放射增敏作用及其相应机制。
分别采用MTT法、实时荧光定量PCR、流式细胞术、免疫荧光、蛋白质免疫印迹、JC - 1染色和Hoechst 33258染色检测细胞增殖、端粒酶活性、细胞周期分布、DNA损伤与修复、p53表达、线粒体膜电位和细胞凋亡。
MST - 312能有效抑制端粒酶活性，在4 μM浓度下对HepG2细胞显示出相对较弱的毒性。与单纯辐射相比，4 μM MST - 312预处理后再进行X射线处理，显著降低了克隆形成能力。观察到DNA损伤加重和亚G1期细胞比例增加。进一步研究发现，同源重组（HR）修复蛋白Rad51焦点核形成受阻，p53表达升高。这些导致线粒体膜电位崩溃，并提高了凋亡率。
这些数据表明，端粒酶功能紊乱可通过损害HR修复过程增强HepG2细胞对X射线辐射的放射敏感性。此外，端粒酶抑制剂MST - 312可能作为联合辐射的辅助治疗药物。
