Laboratory of Pathology, Hebei Medical University, Shijiazhuang, China.
Department of Pathology, The Second Hospital, Hebei Medical University, Shijiazhuang, China.
Toxicol Lett. 2014 Apr 21;226(2):214-21. doi: 10.1016/j.toxlet.2014.02.002. Epub 2014 Feb 10.
Ochratoxin A (OTA), a mycotoxin produced by ubiquitous Aspergilli, is carcinogenic, teratogenic, and nephrotoxic in both humans and animals. Our previous study found that OTA induced DNA double-strand breaks (DSBs) and resulted in G2 phase arrest in human gastric epithelium immortalized (GES-1) cells. DSBs can cause genomic instability, mutations, and neoplastic transformations, and improper repair of DSBs may lead to the development of cancer. Rad51 is a key protein in the homologous recombination (HR) pathway of DSBs repair. The roles of Rad51 in the repair of DNA damage vary in response to different types of cytotoxic agents. The effect of OTA on Rad51 expression and its putative role in the OTA-induced DSBs in GES-1 cells are still not clear enough. The aim of the current study is to elucidate the role of Rad51 in OTA-induced DSBs in GES-1 cells. The results showed that OTA treatment decreased Rad51 expression in a dose- and time-dependent manner. Specific downregulation of Rad51 by siRNA induced DSBs and G2 phase arrest. Rad51 overexpression by transfection with a Rad51-expressing plasmid partly rescued the DSBs and G2 phase arrest in OTA-treated cells. The findings indicate that downregulation of Rad51 contributes to OTA-induced DNA damage in GES-1 cells. Knockdown of p53 with siRNA for 48h effectively reversed the downregulation of Rad51, and decreased the OTA-induced DSBs in GES-1 cells. In addition, the downregulation of Rad51 induced by OTA could be significantly attenuated with specific ERK inhibitor PD98059 or specific p38 MAPK inhibitor SB203580 pre-treatment in GES-1 cells. Thus, the results suggest that downregulation of Rad51 participates in OTA-induced DNA double-strand breaks in GES-1 cells in vitro. And p53, ERK and p38 signaling pathways are all involved in the process.
赭曲霉毒素 A（OTA）是一种普遍存在的曲霉产生的真菌毒素，对人类和动物具有致癌性、致畸性和肾毒性。我们之前的研究发现，OTA 诱导 DNA 双链断裂（DSB），并导致人胃上皮永生化（GES-1）细胞停滞在 G2 期。DSB 可导致基因组不稳定、突变和肿瘤转化，而 DSB 的不当修复可能导致癌症的发展。Rad51 是 DSB 修复同源重组（HR）途径中的关键蛋白。Rad51 在不同类型细胞毒性剂诱导的 DNA 损伤修复中的作用因细胞类型而异。OTA 对 Rad51 表达的影响及其在 GES-1 细胞中 OTA 诱导的 DSB 中的潜在作用尚不清楚。本研究旨在阐明 Rad51 在 GES-1 细胞中 OTA 诱导的 DSB 中的作用。结果表明，OTA 处理以剂量和时间依赖的方式降低 Rad51 的表达。Rad51 的特异性下调通过 siRNA 诱导 DSB 和 G2 期阻滞。用 Rad51 表达质粒转染过表达 Rad51 部分挽救了 OTA 处理细胞中的 DSB 和 G2 期阻滞。研究结果表明，Rad51 的下调导致 GES-1 细胞中 OTA 诱导的 DNA 损伤。用 siRNA 敲低 p53 48h 可有效逆转 Rad51 的下调，并减少 GES-1 细胞中 OTA 诱导的 DSB。此外，在 GES-1 细胞中，用特异性 ERK 抑制剂 PD98059 或特异性 p38 MAPK 抑制剂 SB203580 预处理可显著减弱 OTA 诱导的 Rad51 下调。因此，研究结果表明，Rad51 的下调参与了体外 GES-1 细胞中 OTA 诱导的 DNA 双链断裂。并且 p53、ERK 和 p38 信号通路都参与了这一过程。
