Gastric Epithelial Reactive Oxygen Species Prevent Normoxic Degradation of Hypoxia-inducible Factor-1 in Gastric Cancer Cells

The expression of hypoxia inducible factor (HIF)-1 protein is tightly regulated by cellular oxygen status. Namely, HIF-1 protein is degraded rapidly in normoxic cells, whereas hypoxia stabilizes HIF-1 to transactivate hypoxia-responsive genes. Here we show that HIF-1 protein is expressed aberrantly in gastric cancer cells under normoxia in a reactive oxygen species (ROS)-dependent manner. The normoxic expression of HIF-1 in concordance with its DNA binding activity enhances the transcription of target genes such as vascular endothelial growth factor. The aberrant normoxic expression of HIF-1 is not associated with genetic abnormalities such as the loss of von Hippel-Lindau tumor suppressor, but is well correlated with endogenous ROS (hydrogen peroxide) generation. HIF-1 expression is blocked by nonmitochondrial ROS inhibitors, but not by inhibitors of mitochondrial electron transfer, which indicates that nonmitochondrial ROS stabilize HIF-1 protein in these cells. Gastric epithelial ROS have been linked to Helicobacter pylori-induced gastric carcinogenesis. This study demonstrates for the first time that ROS from H. pylori-infected gastric epithelial cells induce HIF-1 expression and subsequently activate HIF-1 -mediated transcription. Taken together, these results provide a novel mechanism of HIF-1 stabilization in gastric cancer, and demonstrate that gastric epithelial ROS, endogenously generated or H. pylori-stimulated, lead to the constant expression of HIF-1 protein under normoxia. INTRODUCTION HIF-1 is a transcription factor, composed of HIF-1 and (1), which transactivates crucial target genes involved in angiogenesis, energy metabolism, and cell proliferation (2–5). Whereas HIF-1 is constitutively expressed, HIF-1 is tightly regulated in an oxygen-dependent manner (6–9). Under normoxia, HIF-1 undergoes rapid degradation, which is dependent on post-translational modification of HIF-1 . If HIF-1 is hydroxylated at its proline residues of its oxygen-dependent degradation domain under normoxic condition, it binds with the pVHL-containing complex, which targets HIF-1 for ubiquitinproteasome degradation (10–14). When cells are exposed to hypoxia, proline is not hydroxylated and, thus, HIF-1 escapes degradation, allowing it to accumulate and transactivate hypoxia-responsive genes. The concept that hypoxia plays a major role in HIF-1 regulation has been evolving. In addition to hypoxia, it has been demonstrated that the stabilization of HIF-1 protein is induced by genetic abnormalities, such as the loss of pVHL in renal cancer cells like RCC9 (10) or by an activation of PI3K and Akt in prostate cancer cells such as PC3 (15). In addition, the cellular redox status also affects the expression of HIF-1 protein. It is known that hypoxic ROS are associated with the induction of HIF-1 expression in Hep3B hepatoma cells (16, 17). However, nonhypoxic ROS tend to degrade HIF-1 protein in alveolar epithelial cells (18). Thus, the effect of ROS on HIF-1 protein might be determined by oxygen status or cell type. Compared with normal cells, cancer cells have increased metabolism and generate ROS, which has diverse effects on cell survival or cell death (19–24). In gastric epithelial cells, Helicobacter pylori is considered to be a risk factor for gastric cancer. H. pylori-infected gastric epithelial cells generate ROS, which plays an important role in gastric carcinogenesis (25–29). However, the molecular mechanisms by which epithelial ROS is involved in gastric carcinogenesis are unclear. Furthermore, the physiological function of gastric epithelial ROS in gastric cancer is poorly understood. In the present study, we investigated the expression of HIF-1 protein and the underlying mechanism of its stabilization in gastric cancer under normoxic conditions. Our data provide a novel mechanism of HIF-1 stabilization under normoxia, namely that gastric epithelial ROS protect HIF-1 from normoxic degradation in gastric cancer. Received 5/15/02; revised 7/26/02; accepted 8/22/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant 05-2001-001 from the Seoul National University Hospital Research Fund (to T-Y. K. and J-H. P.), Grant CRI-01-04 from the Cancer Research Institute (to T-Y. K. and J-H. P.), Seoul National University College of Medicine, and 2001 BK 21 Project for Medicine, Dentistry and Pharmacy (to T-Y. K., J-H. P., H-S. J., T. Y. K., and Y-J. B.). 2 These authors contributed equally to the work. 3 To whom requests for reprints should be addressed, at Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-799, Korea. Phone: 82-2-760-2390; Fax: 82-2-762-9662; E-mail: [email protected]. 433 Vol. 9, 433–440, January 2003 Clinical Cancer Research Cancer Research. on October 31, 2017. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from MATERIALS AND METHODS Cells and Chemicals. Human gastric adenocarcinoma cells (SNU-484, 601, 638, 668, and 719) were obtained from the Korean Cell Line Bank (30) and grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FBS and gentamicin (10 g/ml). Human hepatoma cells (HepG2) and prostate cancer cells (PC-3) were obtained from the American Type Culture Collection and grown in RPMI 1640 or DMEM supplemented with 10% FBS and gentamicin (10 g/ml). All of the cells were incubated under standard culture condition (20% O2 and 5% CO2, at 37°C). MG132 (Sigma) was used at a final concentration of 10 M. ROS inhibitors were purchased from Sigma. DPI and PDTC were both treated at concentrations of 10, 50, and 100 M. NAC was treated at 1, 10, and 20 mM. Inhibitors of the mitochondrial electron-transfer system, such as rotenone, amobarbital, antimycin A, and KCN, were used at concentrations of 10 g/ml, 10 mM, 10 g/ml, and 10 mM,