Anti-NOX1 (aa 242-271) polyclonal antibody

BACKGROUND & AIMS: Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. METHODS: Fourteen-day-old Nox1-/-mice, Nox4-/mice, Nox1-/-Nox4-/-(double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1DHep), hepatic stellate cells (Nox1DHep), or macrophages (Nox1DMac). Some mice were also given injections of the NOX1specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. RESULTS: Nox4-/-mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/-mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1DHep and Nox1DHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1DMac developed fewer and smaller tumors, similar to Nox1-/-mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/-or Nox1DMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1DMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. CONCLUSIONS: In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damageassociated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.

Chronic non-communicable diseases share the pathomechanism of increased reactive oxygen species (ROS) production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, known as Nox. The recent discovery that expression of Nox1, a Nox isoform that has been implicated in the pathogenesis of cardiovascular and kidney disease and cancer is regulated by the expression and activity of G protein-coupled estrogen receptor (GPER) led to the identification of orally active small-molecule GPER blockers as selective Noxi downregulators (NDRs). Preclinical studies using NDRs have demonstrated beneficial effects in vascular disease, hypertension, and glomerular renal injury. These findings suggest the therapeutic potential of NDRs, which reduce Nox1 protein levels, not only for cardiovascular disease conditions including arterial hypertension, pulmonary hypertension, heart failure with preserved ejection fraction (HFpEF), and chronic renal disease, but also for other non-communicable diseases, such as cerebrovascular disease and vascular dementia, Alzheimer's disease, autoimmune diseases and cancer, in which elevated Nox1-derived ROS production plays a causal role.