BACKGROUND Arsenic 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. METHODS Gastric 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. RESULTS We 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. CONCLUSION These 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.

中文翻译：

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硫化砷通过抑制胃癌细胞中的NFATc3诱导RAG1依赖性DNA损伤，从而杀死细胞。

背景技术发现硫化砷具有潜在的抗癌活性，尤其是在胃癌中。但是，需要进一步探索其潜在机制。本研究旨在探讨砷化合物对胃癌的作用机制。方法用携带shNFATc3的慢病毒载体感染胃癌细胞和/或用硫化砷处理。进行MTT测定以评估细胞生长。流式细胞仪检测用于检测胃癌细胞的细胞周期和活性氧（ROS）水平。进行了Western blot检测胃癌细胞株中活化的T细胞的核因子，细胞质3（NFATc3），细胞周期标志物，DNA损伤途径蛋白表达以及其他蛋白表达。通过RNA测序分析和实时定量PCR确定重组激活基因1（RAG1）在胃癌细胞系中的表达。通过CHIP-qPCR分析确定NFATc3对RAG1的作用。体内评估了砷化砷对AGS细胞的作用。结果我们表明，通过增加免疫球蛋白V（D）J重组所必需的内切核酸酶RAG1的表达，硫化砷以及NFATc3的敲低导致胃癌细胞中双链DNA损伤的增加。NFATc3的过表达阻止了RAG1的表达和硫化砷诱导的DNA损伤。硫化砷诱导细胞氧化应激以重新分布NFATc3，从而抑制其转录功能，这种转录功能可以被N-乙酰基-L-半胱氨酸（NAC）逆转。我们显示NFATc3靶向RAG1的启动子进行转录抑制。我们进一步显示，NFATc3上调和RAG1上调与胃癌患者预后不良相关。我们的体内实验进一步证实，硫化砷通过抑制NFATc3激活RAG1途径发挥了对胃癌细胞的细胞毒活性。结论这些结果表明，硫化砷靶向NFATc3以诱导双链DNA断裂（DSB）通过激活RAG1表达来杀死细胞。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系起来，以此作为其细胞毒性作用的机制。我们进一步显示，NFATc3上调和RAG1上调与胃癌患者预后不良相关。我们的体内实验进一步证实，硫化砷通过抑制NFATc3激活RAG1途径发挥了对胃癌细胞的细胞毒活性。结论这些结果表明，硫化砷靶向NFATc3以诱导双链DNA断裂（DSB）通过激活RAG1表达来杀死细胞。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系起来，以此作为其细胞毒性作用的机制。我们进一步显示，NFATc3上调和RAG1上调与胃癌患者预后不良相关。我们的体内实验进一步证实，硫化砷通过抑制NFATc3激活RAG1途径发挥了对胃癌细胞的细胞毒活性。结论这些结果表明，硫化砷靶向NFATc3以诱导双链DNA断裂（DSB）通过激活RAG1表达来杀死细胞。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系起来，以此作为其细胞毒性作用的机制。结论这些结果表明，硫化砷靶向NFATc3以诱导双链DNA断裂（DSB）通过激活RAG1表达来杀死细胞。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系起来，以此作为其细胞毒性作用的机制。结论这些结果表明，硫化砷靶向NFATc3以诱导双链DNA断裂（DSB）通过激活RAG1表达来杀死细胞。我们的结果将砷化合物与DNA损伤控制和RAG1表达的调节联系起来，以此作为其细胞毒性作用的机制。