Zhang Zhe-Wei, Xiao Jing, Luo Wei, Wang Bo-Han, Chen Ji-Min
Department of Urology, Second Affiliated Hospital; Ministry of Education Key Laboratory of Cancer Prevention and Intervention, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China.
Chin Med J (Engl). 2015 Nov 5;128(21):2938-45. doi: 10.4103/0366-6999.168065.
Caffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated (ATM) activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effects of caffeine on DNA damage responses in cells from the bladder cancer cell line RT4 those were exposed to ionizing radiation (IR).
Immunofluorescent staining was performed to investigate changes in the proteins involved in DNA damage responses with or without caffeine. A mouse xenograft model was used to study the effects of caffeine on the DNA damage responses. Western blotting was used to investigate the effects of caffeine pretreatment on the ATM-Chk2-p53-Puma axis, while real-time polymerase chain reaction (RT-PCR) assessed changes in messenger RNA levels of p53 and downstream targets responding to IR. Finally, terminal deoxynucleotidyl transferase-dUTP nick end labeling assay. Western blotting and colony formation assay were used to measure the effects of caffeine on radiation-related apoptosis. All of the data were analyzed with a two-tailed Student's t-test.
Immunofluorescent staining showed that caffeine pretreatment profoundly suppressed the formation of γH2AXand p53-binding protein 1 foci in RT4 cells in response to irradiation. Cellular and animal experiments suggested that this suppression was mediated by suppression of the ATM-Chk2-p53-Puma DNA damage-signaling axis. RT-PCR indicated caffeine also attenuated transactivation of p53 and p53-inducible genes. The colony formation assay revealed that caffeine displayed radioprotective effects on RT4 cells in response to low-dose radiation compared to the radiosensitization effects on T24 cells.
Caffeine may inhibit IR-related apoptosis of bladder cancer RT4 cells by suppressing activation of the ATM-Chk2-p53-Puma axis.
咖啡因可抑制共济失调毛细血管扩张症及Rad3相关蛋白(ATR)和共济失调毛细血管扩张症突变蛋白(ATM)的活性;ATM是DNA损伤检测的主要激酶。本研究旨在探讨咖啡因对膀胱癌细胞系RT4细胞经电离辐射(IR)后DNA损伤反应的影响。
进行免疫荧光染色以研究有无咖啡因时DNA损伤反应相关蛋白的变化。使用小鼠异种移植模型研究咖啡因对DNA损伤反应的影响。蛋白质印迹法用于研究咖啡因预处理对ATM-Chk2-p53-Puma轴的影响,而实时聚合酶链反应(RT-PCR)评估p53及其对IR反应的下游靶点信使RNA水平的变化。最后,采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法、蛋白质印迹法和集落形成试验来检测咖啡因对辐射相关凋亡的影响。所有数据均采用双侧Student's t检验进行分析。
免疫荧光染色显示,咖啡因预处理可显著抑制RT4细胞经照射后γH2AX和p53结合蛋白1病灶的形成。细胞和动物实验表明,这种抑制是由ATM-Chk2-p53-Puma DNA损伤信号轴的抑制介导的。RT-PCR表明咖啡因还减弱了p53及其诱导基因的反式激活。集落形成试验显示,与对T24细胞的放射增敏作用相比,咖啡因对RT4细胞在低剂量辐射下具有辐射保护作用。
咖啡因可能通过抑制ATM-Chk2-p53-Puma轴的激活来抑制膀胱癌RT4细胞的IR相关凋亡。