Therapeutic effects of the PKR inhibitor C16 suppressing tumor proliferation and angiogenesis in hepatocellular carcinoma in vitro and in vivo

Abstract

The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems

“…Next, we tested the possible effect of other two viral dsRNA‐mediated innate antiviral mechanisms on shaping the transient phenotype of IVT‐CoV2‐Rep, specifically the PKR and oligoadenylate synthetase‐RNase L. 47 , 48 We first used compound C16, an inhibitor of PKR, 49 , 50 to treat the CHO‐K1 cells transfected with IVT‐CoV2‐Rep mRNA. The result demonstrated that C16 treatment increased replicon replication at day 1 posttransfection compared to DMSO control, but this pro‐viral effect disappeared quickly by Day 2 (Figure 5C ).…”

“…[37][38][39][40] In light of the fact that IVT-CoV2-Rep does not encode viral structural and accessory proteins, we speculated that the transfected replicon RNA and/or replication intermediates might induce cellular innate immunity which eliminates replicon replication. However, it is also worth noting that among the above tested host cell lines, Huh7.5 possesses a mutationally inactivated RIG-I for IFN production, 41 Vero cells lack the IFN-β gene, 42,43 and BHK-21 cells have a compromised IFN induction and response, [44][45][46] 47,48 We first used compound C16, an inhibitor of PKR, 49,50 to treat the CHO-K1 cells transfected with IVT-CoV2-Rep mRNA. The result demonstrated that C16 treatment increased replicon replication at day 1 posttransfection compared to DMSO control, but this pro-viral effect disappeared quickly by Day 2 (Figure 5C).…”

Double-Stranded RNA Dependent Protein Kinase (PKR) Deficiency Protects the Liver Against Ischemia/reperfusion Injury

“…C16 is a small molecule that can suppress the activation of PKR and prevents the translational block that follows PKR exposure (Jammi et al 2003, Shimazawa et al 2006). C16 has been extensively used to determine the role of PKR both in vivo (Xiao et al 2016) and in vitro (Watanabe et al 2020). The dose of C16 in this study was based on previous experience in which mice received C16 i.p.…”

Nucleic Acid-Induced Signaling in Chronic Viral Liver Disease

“…Among the nucleic acid sensors with direct antiviral action, PKR and ADAR have been implicated in HCC pathogenesis (142)(143)(144). A PKR inhibitor (C16) reduced cell division in Huh7 cells and markedly decreased liver tumor growth in a mouse model.…”

“…Moreover, it occurred that microvessel density of such tumors was decreased during PKR inhibition. Indeed, in vitro experiments revealed that angiogenesis-relevant growth factor expression-i.e., vascular endothelial growth factor A (VEGF-A), VEGF-B, platelet-derived growth factor A (PDGF-A), PDGF-B, fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and hepatocyte growth factor (HGF)-was significantly downregulated by PKR inhibition (142). These observations are in line with the results reported following analysis of a human HCC cohort (100% HCV-associated), showing increased activity of PKR in tumor samples as compared to cirrhotic tissues (143).…”