Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach

Abstract

Helicobacter pylori infection has been suggested to stimulate expression of the NADPH oxidase 1 (Nox1)-based oxidase system in guinea pig gastric epithelium, whereas Nox1 mRNA expression has not yet been documented in the human stomach. PCR of human stomach cDNA libraries showed that Nox1 and Nox organizer 1 (NOXO1) messages were absent from normal stomachs, while they were specifically coexpressed in intestinal- and diffuse-type adenocarcinomas including signet-ring cell carcinoma. Immunohistochemistry showed that Nox1 and NOXO1 proteins were absent from chronic atrophic gastritis (15 cases), adenomas (4 cases), or surrounding tissues of adenocarcinomas (45 cases). In contrast, Nox1 and its partner proteins were expressed in intestinal-type adenocarcinomas (19/21 cases), diffuse-type adenocarcinomas (15/15 cases), and signet-ring cell carcinomas (9/9 cases). Confocal microscopy revealed that Nox1, NOXO1, Nox activator 1, and p22^phox were predominantly associated with Golgi apparatus in these cancer cells, while diffuse-type adenocarcinomas also contained cancer cells having Nox1 and its partner proteins in their nuclei. Nox1-expressing cancer cells exhibited both gastric and intestinal phenotypes, as assessed by expression of mucin core polypeptides. Thus, the Nox1-base oxidase may be a potential marker of neoplastic transformation and play an important role in oxygen radical- and inflammation-dependent carcinogenesis in the human stomach.

Introduction

Recently, as potential cellular sources of reactive oxygen species (ROS), six homologues of the catalytic core of phagocyte NADPH oxidase (gp91^phox) have been identified in nonphagocytic cells and termed as the NADPH oxidase (Nox)/dual oxidase (Duox) family [1], [2], [3]. Among them, Nox1 was the first identified homologue of gp91^phox (now termed Nox2) [4]. Nox1-derived ROS have been suggested to regulate local innate immunity, inflammation, cell growth, and vascular tone, and to possibly contribute to carcinogenesis [5]. NIH–3T3 cells overexpressing Nox1 enhanced generation of superoxide anion (O₂⁻) and hydrogen peroxide, possibly contributing to tumorigenic and angiogenic functions both in vitro and in vivo [4], [6], [7]. However, a subsequent study revealed that the Nox1-transfected NIH–3T3 cells also carried a mutation of Ras, which may also have accounted for their abnormal growth and transformation [2]. On the other hand, Ras oncogenes up-regulate the expression of Nox1, and Nox1-derived ROS are suggested to participate in oncogenic Ras transformation [8]. In addition, increased cellular ROS generation is linked to the enhanced growth stimulated by growth factors [9], [10], and Nox1 has been suggested to participate in this growth factor-triggered ROS production [11]. Thus, there is still controversy regarding the mechanism of the potential role of Nox1 in carcinogenesis.

The Nox2-based phagocyte oxidase system consists of the membrane-integrated flavocytochrome b₅₅₈, composed of Nox2 and p22^phox, and four cytosolic components (p47^phox, p67^phox, p40^phox, and Rac) that associate with the flavocytochrome to form an active enzyme [12]. Several lines of recent evidence have reveled that the Nox1-based oxidase is also a multicomponent enzyme system. Nox1 is associated with the membrane-integrated protein p22^phox to form a functional heterodimer [13], [14], [15] and requires at least two additional cofactors, Nox organizer 1 (NOXO1) and Nox1 activator 1 (NOXA1) [16], [17], [18]. NOXO1 is likely to act as a “Nox organizer” that bridges interactions between p22^phox and NOXA1. NOXA1 is a homologue of p67^phox that binds to Rac1 or Rac2 and promotes electron flow through the flavocytochrome in a GTP-dependent manner [19], [20]. Rac1 is also suggested to participate in the activation of the multicomponent Nox1-based oxidase [11], [21], [22], [23], [24]. Thus, these partner proteins in addition to Nox1 itself must be examined to elucidate the pathophysiological implications of ROS derived from Nox1.

Gastric cancer is associated with chronic inflammation caused by Helicobacter pylori (H. pylori) infection [28], [29], [30], and the concept of the multistep progression of gastric cancer suggests an important role of ROS particularly in the course of transformation into dysplasia and cancer [30], [31], [32]. Guinea pig gastric mucosal cells in culture constitutively express Nox1, p22^phox, and NOXA1 [21]. They produce high levels of O₂⁻ when exposed to H. pylori lipopolysaccharide (LPS) [25], [26]. ROS derived from the cells stimulate cell growth, inhibit apoptosis of aged cells, and additionally enhance Toll-like receptor 4-mediated activation of nuclear factor-κB and production of inflammatory cytokines [27]. A subsequent study revealed that H. pylori LPS stimulated the transcription of Nox1 and NOXO1 genes, and activated Rac1 [21]. These results postulated that H. pylori infection might stimulate the expression of Nox1 and NOXO1, facilitating ROS-dependent carcinogenesis. On the other hand, the expression of Nox1 message has been systemically examined in various human tissues [4], [33], [34] and also in the stomach [18], [35], [36], whereas the expression of Nox1 message has not yet been documented in the human stomach. One might say that expression of Nox1 in the stomach is species specific [5]. Thus, the understanding of the role of Nox1 in the human stomach is still at the preliminary stage.

To address these issues, we precisely examined cellular and subcellular localization of Nox1 and its partner proteins in the human stomach, and found that a Nox1-based oxidase system was specifically expressed in both intestinal- and diffuse-type adenocarcinomas of the human stomach. Our results implicate that dysregulated expression of the Nox1-based oxidase system may be associated with inflammation- and ROS-dependent carcinogenesis in the human stomach.