Arsenic sulfide induces RAG1-dependent DNA damage for cell killing by inhibiting NFATc3 in gastric cancer cells
Arsenic sulfide induces RAG1-dependent DNA damage for cell killing by inhibiting NFATc3 in gastric cancer cells
复制标题
硫化砷通过抑制胃癌细胞中的 NFATc3 诱导 RAG1 依赖性 DNA 损伤，从而实现细胞杀伤
DOI：
10.1186/s13046-019-1471-x
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发表时间：
2019-12-10
影响因子：
11.3
通讯作者：
Chen, Siyu
中科院分区：
文献类型：
--
作者：
Kang, Ting;Ge, Maolin;Chen, Siyu
研究背景硫化亚砷被发现具有潜在的抗癌活性，特别是在胃癌中。然而，其背后的机制还需要进一步探索。方法用携带shNFATc3的慢病毒载体感染人胃癌细胞株和/或用硫化砷处理胃癌细胞。用四甲基偶氮唑盐比色法检测细胞生长情况。采用流式细胞仪检测细胞周期和细胞内活性氧(ROS)水平。Western印迹法检测活化T细胞核因子、胞浆3(NFATc3)、细胞周期标志物、DNA损伤途径蛋白表达及其他蛋白的表达。采用RNA测序和实时定量定量聚合酶链式反应检测重组激活基因1(RAG1)在胃癌细胞系中的表达。采用芯片定量聚合酶链式反应方法检测NFATc3对RAG1的影响。结果硫化砷和NFATc3基因敲除可通过上调免疫球蛋白V(D)J重组所必需的核酸内切酶RAG1的表达，导致胃癌细胞双链DNA损伤增加。NFATc3过表达可阻断硫化砷诱导的RAG1表达和DNA损伤。硫化砷诱导细胞氧化应激重新分布NFATc3，从而抑制其转录功能，而N-乙酰-L-半胱氨酸(NAc)可逆转这一作用。我们发现NFATc3针对RAG1的启动子进行转录抑制。我们进一步表明，NFATc3的上调和RAG1的下调与胃癌患者的不良预后显著相关。我们的体内实验进一步证实了硫化砷通过抑制NFATc3激活RAG1通路而发挥对胃癌细胞的杀伤作用。结论硫化砷靶向NFATc3通过激活RAG1表达诱导双链DNA断裂(DSB)，从而达到杀伤细胞的目的。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系在一起，作为其细胞毒作用的机制。
BackgroundArsenic sulfide was found to have potential anti-cancer activities, especially in gastric cancer. However, the underlying mechanism need to be further explored. This study was aimed to investigate the mechanism of arsenic compounds on gastric cancer.MethodsGastric cancer cell lines were infected with lentiviral vector carrying shNFATc3 and/or treated with arsenic sulfide. MTT assay were performed to assess cell growth. Flow cytometer assays were used to detect cell cycle and reactive oxygen species (ROS) level of gastric cancer cells. Western blot was carried out to detect nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3), cell cycle markers, DNA damage pathway protein expression as well as other protein expression in gastric cancer cell lines. The expression of recombination activating gene 1 (RAG1) in gastric cancer cell lines was determined by RNA-sequencing analyses and Real-Time qPCR. The effect of NFATc3 on RAG1 were determined by CHIP-qPCR assay. The effect of arsenic sulfide on AGS cells was evaluated in vivo.ResultsWe show that arsenic sulfide as well as knockdown of NFATc3 resulted in increased double-strand DNA damage in gastric cancer cells by increasing the expression of RAG1, an endonuclease essential for immunoglobulin V(D) J recombination. Overexpression of NFATc3 blocked the expression of RAG1 expression and DNA damage induced by arsenic sulfide. Arsenic sulfide induced cellular oxidative stress to redistribute NFATc3, thereby inhibiting its transcriptional function, which can be reversed by N-acetyl-L-cysteine (NAC). We show that NFATc3 targets the promoter of RAG1 for transcriptional inhibition. We further showed that NFATc3 upregulation and RAG1 downregulation significantly associated with poor prognosis in patients with gastric cancer. Our in vivo experiments further confirmed that arsenic sulfide exerted cytotoxic activity against gastric cancer cells through inhibiting NFATc3 to activate RAG1 pathway.ConclusionThese results demonstrate that arsenic sulfide targets NFATc3 to induce double strand DNA break (DSB) for cell killing through activating RAG1 expression. Our results link arsenic compound to the regulation of DNA damage control and RAG1 expression as a mechanism for its cytotoxic effect.