Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, United Kingdom.
Radiother Oncol. 2013 Sep;108(3):429-33. doi: 10.1016/j.radonc.2013.06.021. Epub 2013 Aug 6.
In muscle-invasive bladder cancer there is an urgent need to identify relatively non-toxic radiosensitising agents for use in elderly patients. Histone deacetylase inhibitors radiosensitise tumour cells but not normal cells in vitro and variously downregulate DNA damage signalling, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair proteins. We investigated panobinostat (PAN) as a potential radiosensitiser in bladder cancer cells.
Clonogenic assays were performed in RT112 bladder cancer cells, and RT112 cells stably knocked down for RAD51 or Ku80 by shRNAi. Resolution of γH2AX foci was determined by immunofluorescence confocal microscopy, cell cycle progression by FACS analysis and protein expression by western blotting.
PAN had a greater radiosensitising effect in Ku80KD than RT112 or RAD51KD cells; enhancement ratios 1.35 for Ku80KD at 10nM (IC(20) for Ku80KD) and 1.31 for RT112 and RAD51KD at 25 nM (IC(40) for both). PAN downregulated MRE11, NBS1 and RAD51, but not Ku70 and Ku80, increased γH2AX foci formation in a dose-dependent manner and delayed γH2AX foci repair after ionising radiation.
PAN acts as a radiosensitiser in bladder cancer cell lines, and appears to target HR rather than NHEJ. As muscle-invasive bladder tumours have reduced Ku-DNA binding, PAN could be particularly useful as a radiosensitiser in bladder cancer.
在肌层浸润性膀胱癌中，迫切需要寻找相对无毒的放射增敏剂，用于老年患者。组蛋白去乙酰化酶抑制剂在体外能增敏肿瘤细胞而不增敏正常细胞，并能下调 DNA 损伤信号、同源重组（HR）和非同源末端连接（NHEJ）修复蛋白。我们研究了泛素抑制剂（PAN）作为膀胱癌细胞潜在的放射增敏剂。
在 RT112 膀胱癌细胞中进行集落形成实验，并通过 shRNAi 稳定敲低 RAD51 或 Ku80 的 RT112 细胞。通过免疫荧光共聚焦显微镜测定 γH2AX 焦点的分辨率，通过 FACS 分析测定细胞周期进程，通过 Western blot 测定蛋白表达。
PAN 在 Ku80KD 细胞中的放射增敏作用大于 RT112 或 RAD51KD 细胞；在 10 nM（Ku80KD 的 IC（20））时增强比为 1.35，在 25 nM 时增强比为 1.31（RT112 和 RAD51KD 的 IC（40））。PAN 下调了 MRE11、NBS1 和 RAD51，但不包括 Ku70 和 Ku80，以剂量依赖的方式增加 γH2AX 焦点的形成，并在电离辐射后延迟 γH2AX 焦点的修复。
PAN 在膀胱癌细胞系中作为放射增敏剂，似乎靶向 HR 而不是 NHEJ。由于肌层浸润性膀胱癌的 Ku-DNA 结合减少，PAN 可能特别适用于膀胱癌的放射增敏剂。
