NOX1 inhibition attenuates the development of a pro‐tumorigenic environment in experimental hepatocellular carcinoma

Abstract

Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment. Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment. Conclusions NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

The TGF-β/NADPH Oxidases Axis in the Regulation of Liver Cell Biology in Health and Disease

“…DEN-injected wild-type (WT) mice that received a NOX1 inhibitor ML171 [ 174 ] developed fewer and smaller hepatic tumor nodules, compared to their vehicle-treated counterparts [ 38 ]. In agreement, a recent report indicated that pharmacological inhibition of NOX1 with GKT771 (Genkyotex) during HCC progression in mice attenuate the expression of several inflammatory markers, angiogenesis and fibrosis, therefore reducing the pro-tumorigenic environment [ 146 ]. Despite the opposite role of NOX1 and NOX4 in HCC (the first one promoting and the second one suppressing tumor progression), the fact that most of the liver tumor cells express low levels of NOX4 [ 115 ] facilitates the use of general NOX inhibitors as promising tools in the treatment of liver cancer.…”

“…Recent evidence indicates that NOX1 expression in macrophages mediates tumor promoting activity, through activating a ROS/MEK mechanism responsible of inflammatory cytokines production, thereby promoting the survival and proliferation of oncogene-carrying mutant hepatocytes, which ultimately accelerate HCC development [ 38 ]. Pharmacological inhibition of NOX1 with GKT771 during HCC progression in mice attenuates the expression of several inflammatory markers, angiogenesis and fibrosis, therefore reducing the pro-tumorigenic environment [ 146 ]. Besides NOX1, NOX2-derived ROS are also involved in TLR2-dependent M2 macrophage polarization, supporting tumor growth.…”

The NADPH oxidase NOX4 regulates redox and metabolic homeostasis preventing HCC progression

“…On the contrary, NOX1 mediates autocrine growth and survival of liver tumor cells and anti‐apoptotic signals induced by TGF‐β through the transactivation of the EGF receptor pathway 32,33. Indeed, NOX1 pharmacological inhibition impairs cell growth and enhances TGF‐β‐induced apoptosis,34 and attenuates the development of a pro‐tumorigenic environment in experimental HCC 35. Overall, NOX1 and NOX4 exert opposite roles in the control of liver growth and apoptosis, and their balance may dictate cell fate.…”

The Role of Cancer-Associated Fibroblasts in Hepatocellular Carcinoma and the Value of Traditional Chinese Medicine Treatment

“…Tumor microenvironments are characterized as hypoxia and energy deficiency, which render cancer cells and other stromal cells to go through metabolic reprogramming ( 99 ), so as to meet their needs for increasing biological process of bioenergy, biosynthesis, and redox balance ( 100 ). The activation of CAFs in HCC also requires a large quantity of energy to promote cell proliferation, ECM and cytokine release, and the migration toward certain pro-fibrotic and pro-inflammatory regions ( 101 ). As a consequence, metabolic reprogramming and energy expenditure regulation play key roles in the function change of CAFs during the HCC microenvironment and liver injury.…”