Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair
Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair
复制标题
BET 家族蛋白和 HDAC 抑制的组合通过 RAD51 相关的 DNA 损伤修复协同引发软骨肉瘤细胞凋亡
DOI：
10.2147/cmar.s254412
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发表时间：
2020-01-01
影响因子：
3.3
通讯作者：
Wu, Chunfei
中科院分区：
文献类型：
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作者：
Huan, Songwei;Gui, Tao;Wu, Chunfei
背景软骨肉瘤是第二常见的骨肿瘤，对常规化疗具有固有的抵抗力。本研究旨在探讨BET家族蛋白和HDAC抑制剂联合应用对软骨肉瘤的治疗作用及其具体机制。方法用BET家族蛋白抑制剂（JQ 1）和组蛋白去乙酰化酶抑制剂（HDACIs）处理两种软骨肉瘤细胞（伏立诺他/SAHA或帕比司他/PANO）;然后，通过细胞计数试剂盒-8（CCK-8）测定法测定细胞活力，并通过Chou方法计算组合指数（CI）;采用5-乙炔基-2 ′-脱氧尿苷（EdU）掺入法和集落形成实验检测细胞增殖，流式细胞仪检测细胞凋亡和活性氧（ROS）水平;免疫印迹法检测caspase-3、Bcl-XL、Bcl-2、γ-H2 AX和RAD 51蛋白表达，彗星试验检测DNA损伤，免疫荧光法观察RAD 51和γ-H2 AX灶。结果JQ 1与SAHA或PANO联合作用可协同抑制软骨肉瘤细胞的生长和集落形成能力。BET和HDAC联合抑制也显著升高了ROS水平，随后激活了切割的caspase-3，并下调了Bcl-2和Bcl-XL。从机制上讲，JQ 1和SAHA的联合治疗引起了许多DNA双链断裂（DSB），如彗星试验所证明的。γ-H2 AX表达和病灶形成的增加也一致地表明在用JQ 1和SAHA共处理后DNA损伤的积累。此外，发现同源重组（HR）DNA修复的关键蛋白RAD 51被深度抑制。相反，RAD 51的异位表达通过抑制切割型半胱天冬酶-3的表达而部分挽救SW 1353细胞凋亡。结论BET和HDAC抑制剂通过抑制软骨肉瘤细胞中RAD 51相关的HR DNA修复，协同抑制细胞生长并诱导细胞凋亡。
Background Chondrosarcoma is the second-most common type of bone tumor and has inherent resistance to conventional chemotherapy. Present study aimed to explore the therapeutic effect and specific mechanism(s) of combination BET family protein and HDAC inhibition in chondrosarcoma. Methods Two chondrosarcoma cells were treated with BET family protein inhibitor (JQ1) and histone deacetylase inhibitors (HDACIs) (vorinostat/SAHA or panobinostat/PANO) separately or in combination; then, the cell viability was determined by Cell Counting Kit-8 (CCK-8) assay, and the combination index (CI) was calculated by the Chou method; cell proliferation was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assay; cell apoptosis and reactive oxygen species (ROS) level were determined by flow cytometry; protein expressions of caspase-3, Bcl-XL, Bcl-2, γ-H2AX, and RAD51 were examined by Immunoblotting; DNA damage was determined by comet assay; RAD51 and γ-H2AX foci were observed by immunofluorescence. Results Combined treatment with JQ1 and SAHA or PANO synergistically suppressed the growth and colony formation ability of the chondrosarcoma cells. Combined BET and HDAC inhibition also significantly elevated the ROS level, followed by the activation of cleaved-caspase-3, and the downregulation of Bcl-2 and Bcl-XL. Mechanistically, combination treatment with JQ1 and SAHA caused numerous DNA double-strand breaks (DSBs), as evidenced by the comet assay. The increase in γ-H2AX expression and foci formation also consistently indicated the accumulation of DNA damage upon cotreatment with JQ1 and SAHA. Furthermore, RAD51, a key protein of homologous recombination (HR) DNA repair, was found to be profoundly suppressed. In contrast, ectopic expression of RAD51 partially rescued SW 1353 cell apoptosis by inhibiting the expression of cleaved-caspase-3. Conclusion Taken together, our results disclose that BET and HDAC inhibition synergistically inhibit cell growth and induce cell apoptosis through a mechanism that involves the suppression of RAD51-related HR DNA repair in chondrosarcoma cells.