Astrid Vandierendonck1, Helena Degroote2,3, Bart Vanderborght1,4, Xavier Verhelst1, Anja Geerts1, Lindsey Devisscher5, Hans Van Vlierberghe1. 1. Hepatology Research Unit, Department of Internal Medicine and Pediatrics, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Blok B, 2nd floor, 9000, Ghent, Belgium. 2. Hepatology Research Unit, Department of Internal Medicine and Pediatrics, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Blok B, 2nd floor, 9000, Ghent, Belgium. Helena.degroote@ugent.be. 3. Gut-Liver Immunopharmacology Unit, Department of Basic and Applied Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Entrance 36 - Floor 3, 9000, Ghent, Belgium. Helena.degroote@ugent.be. 4. Gut-Liver Immunopharmacology Unit, Department of Basic and Applied Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Entrance 36 - Floor 3, 9000, Ghent, Belgium. 5. Gut-Liver Immunopharmacology Unit, Department of Basic and Applied Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, Entrance 36 - Floor 3, 9000, Ghent, Belgium. lindsey.devisscher@ugent.be.
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.
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 HCCpatients. 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 murineHepa1-6 HCC cell lines, the humanTHP1 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 humanHCC 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 HCCmice 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.
Authors: Samuele De Minicis; Ekihiro Seki; Yong-Han Paik; Christoph H Osterreicher; Yuzo Kodama; Johannes Kluwe; Luciano Torozzi; Katsumi Miyai; Antonio Benedetti; Robert F Schwabe; David A Brenner Journal: Hepatology Date: 2010-10 Impact factor: 17.425
Authors: Femke Heindryckx; Koen Mertens; Nicolas Charette; Bert Vandeghinste; Christophe Casteleyn; Christophe Van Steenkiste; Dominique Slaets; Louis Libbrecht; Steven Staelens; Peter Starkel; Anja Geerts; Isabelle Colle; Hans Van Vlierberghe Journal: Mol Cancer Date: 2010-08-20 Impact factor: 27.401