Moderate doses of GKT771 treatment do not induce HCC cell cytotoxicity in vitro
To assess the potential cytotoxic effects of NOX1 inhibition in vitro, two human HCC cell lines (Huh7 and Hep3B) were treated for 24 hours with various concentrations of GKT771, ranging from 0-100 µM. Mitochondrial metabolism was examined as an indicator of cell viability using the MTT assay, and was found to be significantly reduced in a concentration dependent manner in both cell lines (Figure 2A & D). No loss of membrane integrity or increase in caspase 3/7 activity could be detected using an LDH and CaspGlo® assay respectively, which indicates the absence of cell lysis or apoptotic cell death of human HCC cells upon treatment with GKT771 at concentrations up to 100 µM (Figure 2B-C & E-F).
Identical analyses following a similar GKT771 treatment regimen in the murine Hepa1-6 HCC cell line revealed no significant alterations in mitochondrial metabolism and caspase 3/7 activity (Figure 2G & I). However, an impaired cellular membrane integrity could be observed at 100 µM, indicating the presence of a moderate toxic effect at the highest concentration (Figure 2H).
NOX1 inhibition attenuates LPS-induced macrophage polarization
Murine bone marrow-derived single cell isolates and THP1 cells were seeded in medium containing M-CSF or PMA respectively, to induce proliferation and differentiation into macrophages. The resulting primary BMDMs and THP1 macrophages were treated with 100 µM of GKT771 with and without LPS-stimulation. MTT and LDH analyses showed a significant enhancement of the mitochondrial metabolic state associated with a disruption of cellular membrane integrity of GKT771-treated BMDMs in both the presence and absence of LPS (Figure 3A). In the human monocyte cell line THP1, GKT771 induced a decrease in cell metabolic activity and increased cytotoxicity, in both the presence and absence of LPS (Figure 3B).
To assess the effect of NOX1 inhibition on the polarization of BMDMs in vitro, the mRNA expression of inflammatory markers (CCL2, IL6, IL1β, TNFα, iNOS, NLRP3 and Caspase 1) and of the immune suppressive marker PDL1 was examined via RT-qPCR upon treatment of these cells with 100 µM of GKT771, with and without LPS. LPS activation of the murine BMDMs resulted in a significant increased expression of the analysed markers which was significantly less pronounced in combination with NOX1 inhibition for CCL2, IL6, IL1β, iNOS, NLRP3 and Caspase 1 (Figure 3C). Our results were confirmed in the human monocyte cell line THP1 for the inflammatory markers CCL2, IL6, IL1β and TNFα (Figure 3D). This shows that NOX1 inhibition is able to attenuate LPS-induced polarization in both murine BMDM and the human monocyte cell line THP1.
NOX1 inhibition attenuates the development of a pro-tumorigenic microenvironment during HCC development
Mice received weekly IP injections of DEN for 25 weeks. At week 15, daily treatment with GKT771 was initiated to evaluate the effect of NOX1 inhibition on tumor development. DEN administration resulted in a mild decrease in body weight and a moderately elevated spleen weight (not significant). As expected, DEN administration induced macroscopically visible tumoral lesions in the liver of both vehicle and GKT771 treated mice, while in the healthy control groups (saline) no lesions were detected. Preventive GKT771 treatment did not significantly alter the total number of tumor lesions in the liver (Figure 4A-B). In line, the expression of two HCC markers, AFP and GP3, was induced in tumor and surrounding liver tissue of both vehicle and GKT771 treated HCC mice (Figure 4C). However, significant induction of hepatic cancer stem cell markers (MDR1, EPCAM, CD44 and CD133) in the tumors and surrounding liver tissue was attenuated upon GKT771 treatment (Figure 4C).
DEN-induced HCC resulted in an induced expression of inflammatory markers IL6, TNFα, IL1β, CCR2, CCL2, Caspase 1 and NLRP3, and of the immune suppressive marker PDL1, compared to healthy control liver tissue (Figure 5). This induction was most explicit and significant in the tumor lesions, but also present in surrounding liver tissue. Preventive administration of GKT771 resulted in a less pronounced induction of these markers, both in tumor lesions and surrounding tissue (Figure 5).
To further evaluate the effect of GKT771 on the progressing tumoral microenvironment, mRNA expression analysis of angiogenic and fibrotic markers was performed. NOX1 inhibition was able to attenuate DEN-induced induction of both angiogenic (CD31, END, iCAM, vCAM) and fibrotic (αSMA, COL1A, TGFβ) markers (Figure 6A-B). Histological analysis of liver fibrosis confirmed this anti-fibrotic effect of NOX1 inhibition early in DEN-induced HCC development (Figure 6C).
Early treatment and twice daily dosing of the NOX1 inhibitor is able to attenuate the progression of advanced HCC
To evaluate the therapeutic potential of NOX1 inhibition in advanced HCC, two treatment regimens were set-up including an early and delayed treatment protocol (Figure 1B). In both treatment regimens, DEN administration induced macroscopically visible tumoral lesions in the liver, while in the saline-treated control group no lesions were detected (Figure 7A-B). Early treatment and twice daily dosing of GKT771 during advanced HCC resulted in reduced HCC lesions compared to no treatment (Figure 7A-B). This was confirmed by less induced expression of the HCC markers AFP and GP3, both in the early (twice daily dosing) and delayed (single daily dosing) treatment group compared to untreated HCC mice (Figure 7C). Assessment of the TME of advanced HCC liver tissue showed induction of markers involved in inflammation (IL1β, TNFα, IL6, CCL2, CCR2), inflammasome activation (Caspase 1, NLRP3, iNOS), immune suppression (PDL1), angiogenesis (END, CD31, iCAM, vCAM) and fibrosis (αSMA, TGFβ, COL1A) compared to healthy control livers (Figure 11-13A, light grey bars). In early twice daily dosed treated HCC mice, a (partial) loss of significant induction of IL1β, TNFα, CCL2, CCR2, Caspase 1 and NLRP3 in the HCC lesions could be observed (Figure 8A). This was also confirmed by multiplex analysis showing reduced CCL2, TNF and IL6 production in NOX1i treated mice. Furthermore, treatment with NOX1 inhibition resulted in downstream inhibited ROS production in both treatment regimen (Figure 8B). Expression of angiogenic marker vCAM was less induced in both the non-cancerous surrounding liver tissue and the HCC lesions of early twice daily treated HCC mice (Figure 9A). Fibrosis was attenuated in both early and delayed GKT771 treated HCC mice, indicated by a (partial) loss of significant induction of COL1A and TGFβ expression in the surrounding liver, HCC lesions and total liver tissue (Figure 9B). Furthermore, both treatment regimens resulted in a reduced Metavir score compared to untreated HCC mice (Figure 9C).