Tian Youjia, Liu Guochao, Wang Hui, Tian Zhujun, Cai Zuchao, Zhang Fengmei, Luo Yue, Wang Shue, Guo Gongshe, Wang Xiaowei, Powell Simon, Feng Zhihui
Department of Occupational Health and Occupational Medicine, The Public Health School, Shandong University, Shandong, Jinan, 250012, China.
The Second Hospital of Shandong University, Shandong, Jinan, China.
DNA Repair (Amst). 2017 Oct;58:1-12. doi: 10.1016/j.dnarep.2017.08.002. Epub 2017 Aug 9.
It was reported that valproic acid (VPA, a histone deacetylase inhibitor) can sensitize cancer cells to hydroxyurea (HU, a ribonucleotide reductase inhibitor) for chemotherapy, although the mechanism of VPA-induced HU sensitization is unclear. In this study, we systematically characterized VPA-induced HU sensitization of breast cancer cells. Multiple breast cancer cell models were employed to investigate whether the safe concentration of 0.5mM VPA and 2mM HU can result in DNA double-strand breaks (DSBs) and impact cell survival. Furthermore, the underlying mechanism was explored through cell biology assays, including clonogenic survival, homologous recombination (HR) activity, immunoblot and immunofluorescence. We found that VPA and HU cooperatively suppressed cancer cell survival. VPA resulted in the accumulation of more DNA double-strand breaks (DSBs) in response to HU-induced replication arrest and was able to block HU-stimulated homologous recombination (HR) through inhibiting the activity of two key HR repair proteins by hyperphosphorylation of replication protein A2 (RPA2-p) and recombinase Rad51. However, apoptosis was not detected under this condition. In addition, the results from the survival fraction in the cells expressing defective RPA2-p showed that VPA disrupted the HU-induced RPA2-p-Rad51-mediated HR pathway. Importantly, these findings were further supported by analyzing primary-culture cells from the tissue of chemical carcinogen (DMBA)-induced breast cancer in rats. Thus, our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing RPA2-p-mediated DNA repair pathway, which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.
据报道，丙戊酸（VPA，一种组蛋白去乙酰化酶抑制剂）可使癌细胞对羟基脲（HU，一种核糖核苷酸还原酶抑制剂）化疗敏感，尽管VPA诱导HU敏感的机制尚不清楚。在本研究中，我们系统地描述了VPA诱导乳腺癌细胞对HU敏感的情况。采用多种乳腺癌细胞模型来研究0.5mM VPA和2mM HU的安全浓度是否会导致DNA双链断裂（DSB）并影响细胞存活。此外，通过细胞生物学试验探索潜在机制，包括克隆形成存活、同源重组（HR）活性、免疫印迹和免疫荧光。我们发现VPA和HU协同抑制癌细胞存活。VPA导致在HU诱导的复制停滞时积累更多的DNA双链断裂（DSB），并能够通过对复制蛋白A2（RPA2-p）和重组酶Rad51进行过度磷酸化来抑制两种关键HR修复蛋白的活性，从而阻断HU刺激的同源重组（HR）。然而，在此条件下未检测到细胞凋亡。此外，在表达缺陷型RPA2-p的细胞中存活分数的结果表明，VPA破坏了HU诱导由RPA2-p-Rad51介导的HR途径。重要的是，通过分析大鼠化学致癌物（DMBA）诱导的乳腺癌组织中的原代培养细胞，这些发现得到了进一步支持。因此，我们的数据表明，VPA和HU通过干扰RPA2-p介导的DNA修复途径协同抑制肿瘤细胞，这为联合化疗药物使乳腺癌细胞敏感提供了一种新方法。
