Department of Medicine, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
J Pharmacol Exp Ther. 2011 Oct;339(1):218-27. doi: 10.1124/jpet.111.182352. Epub 2011 Jul 12.
Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have previously shown that NADPH oxidase NOX5-S generates reactive oxygen species (ROS) when Barrett's metaplastic cells are exposed to acid. Besides metaplastic cells, other H(2)O(2)-producing cells (e.g., inflammatory cells) present in BE mucosa may produce additional ROS, which may also affect metaplastic cells contributing to esophageal tumorigenesis. In this study, we investigate whether exogenous H(2)O(2) stimulates cell proliferation by increasing NOX5-S expression. Low dose (10(-13) M) of H(2)O(2) significantly increased thymidine incorporation, NOX5-S mRNA, and protein expression in a Barrett's EA cell line FLO. H(2)O(2)-induced increase in NOX5-S expression was significantly inhibited by knockdown of nuclear factor (NF)-κB1 p50 with p50 small interfering RNA (siRNA) in EA cell lines FLO and OE33. H(2)O(2) significantly increased p65 phosphorylation and the luciferase activity in FLO cells transfected with a NF-κB activation reporter plasmid pNF-κB-Luc. H(2)O(2)-induced increase in luciferase activity in FLO cells was significantly decreased by knockdown of extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase (MAPK). Overexpression of p50 and p65 remarkably increased the luciferase activity in FLO cells transfected with a NOX5-S reporter plasmid NOX5-LP. In addition, H(2)O(2)-induced thymidine incorporation in FLO cells was significantly decreased by the MAPK kinase 1/2 inhibitor 2'-amino-3'methoxyflavone (PD98059) and ERK2 siRNA but not by ERK1 siRNA. Likewise, H(2)O(2)-induced increase in NOX5-S expression was significantly decreased by ERK2 siRNA in FLO and OE33 cells. We conclude that a low dose of H(2)O(2) increases cell proliferation. H(2)O(2)-induced increase in cell proliferation may depend on sequential activation of ERK2 MAPK, NF-κB1 p50, and NOX5-S.
胃酸反流加速巴雷特食管（BE）向食管腺癌（EA）进展的机制尚不完全清楚。我们之前已经表明，当巴雷特化生细胞暴露于酸时，NADPH 氧化酶 NOX5-S 会产生活性氧（ROS）。除了化生细胞之外，BE 黏膜中存在的其他产生 H 2 O 2 的细胞（例如炎症细胞）也可能产生额外的 ROS，这也可能影响化生细胞，从而促进食管癌变。在这项研究中，我们研究了外源性 H 2 O 2 是否通过增加 NOX5-S 表达来刺激细胞增殖。低剂量（10 -13 M）的 H 2 O 2 显著增加了巴雷特 EA 细胞系 FLO 的胸苷掺入、NOX5-S mRNA 和蛋白表达。用 NF-κB1 p50 的 p50 小干扰 RNA（siRNA）在 EA 细胞系 FLO 和 OE33 中敲低 NF-κB1 p50 显著抑制了 H 2 O 2 诱导的 NOX5-S 表达增加。H 2 O 2 显著增加了转染 NF-κB 激活报告质粒 pNF-κB-Luc 的 FLO 细胞中 p65 的磷酸化和荧光素酶活性。用 MAPK 丝裂原激活蛋白激酶（MAPK）的细胞外信号调节激酶 2（ERK2）抑制剂 2'-氨基-3'-甲氧基黄酮（PD98059）和 ERK2 siRNA 显著降低了 FLO 细胞中荧光素酶活性的增加。过表达 p50 和 p65 显著增加了转染了 NOX5-S 报告质粒 NOX5-LP 的 FLO 细胞中的荧光素酶活性。此外，用 MAPK 激酶 1/2 抑制剂 2'-氨基-3'-甲氧基黄酮（PD98059）和 ERK2 siRNA 显著降低了 FLO 细胞中 H 2 O 2 诱导的胸苷掺入，但不降低 ERK1 siRNA。同样，H 2 O 2 诱导的 NOX5-S 表达增加在 FLO 和 OE33 细胞中也被 ERK2 siRNA 显著降低。我们得出结论，低剂量的 H 2 O 2 可增加细胞增殖。H 2 O 2 诱导的细胞增殖增加可能依赖于 ERK2 MAPK、NF-κB1 p50 和 NOX5-S 的顺序激活。
