In the present study, we identified a phytochemical lucidone with strong inhibitory effects on HCV replication in both HCV replicon and JFH-1-infected cells (). In addition, we found that lucidone treatment resulted in the elevation of cellular HO-1 expression at the transcriptional level (), leading to accumulation of its product biliverdin for anti-HCV activity (). These results are supported by recent findings of Lehmann et al. and Zhu et al. demonstrating that the HO-1 metabolic product biliverdin inhibited viral replication by targeting IFN signaling pathway and viral NS3/4A protease (12
) ( and ). For comparison, lucidone displayed the efficient inhibition of viral replication by as much as 80% at a concentration of 50 μM in both assay systems (), which was similar to or even better than that of bilividin on anti-HCV replication at concentrations of approximately 50 to 200 μM in those earlier reports. Investigation of the mechanism disclosed that HO-1 inhibition by HO-1 inhibitor or shRNA gradually abrogated the anti-HCV activity of lucidone (). Moreover, we observed high accumulation of cellular and nuclear Nrf2 and subsequently confirmed the specific transactivation of HO-1 expression by Nrf2 using an ARE-luciferase activity assay in lucidone-treated cells (). In contrast, depletion of Nrf2 by shRNA resulted in the abrogation of HO-1 induction and anti-HCV activity of lucidone, clearly indicating that the inhibitory effect of lucidone on HCV replication was because of Nrf2-ARE-dependent HO-1 induction (). The proposed model of anti-HCV action of lucidone was illustrated in . Lucidone enhances Nrf2 expression and triggers its nuclear translocation, leading to an activation of HO-1 expression. Subsequently, an accumulation of its metabolite biliverdin then inhibits HCV replication through increasing antiviral interferon response and blocking HCV NS3/4A protease activity.
Model for the inhibitory action of lucidone on HCV replication.
A previous report demonstrated that treatment with miRNA-196, which targets Bach1, a transcriptional repressor of HO-1, resulted in HO-1 upregulation and subsequent suppression of HCV replication (13
). In addition to suppressing HCV replication, the induction or overexpression of HO-1 has been demonstrated to interfere with the replication of other viruses, such as HIV (38
), hepatitis B virus (39
), and enterovirus 71 (40
). Therefore, targeting HO-1 may be a potential therapeutic approach to inhibit virus infection. The present study is the first time to identify a phytocompound as a protective agent against HCV replication via HO-1 induction. In addition to Nrf2, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are also involved in the transactivation of HO-1 expression (10
). We will further investigate the effect of lucidone on the NF-κB and AP-1 expression in terms of HO-1 upregulation. In addition, HO-1 gene expression can be transcriptionally mediated by a number of intracellular signaling molecules, such as extracellular signal-regulated protein kinase (ERK) (41
), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase, protein kinase C, and phosphatidylinositol 3-kinase (8
). Among them, the activation of MAPK/ERK kinase-ERK1/2 signaling has been reported to be involved in anti-HCV activity upon oxidative stress (42
). Moreover, lucidone was previously reported to exert protective effects against LPS-induced inflammation through NF-κB/MAPK signaling pathways (18
). Therefore, it is worthwhile to further investigate additional molecules or signaling pathways involved in HO-1 activation by lucidone to clearly demonstrate the detailed molecular mechanism by which lucidone blocks HCV replication.
A number of new direct-acting antivirals (DAAs) for treating HCV infection are currently under development. Telaprevir, a recently approved NS3/4A protease inhibitor, significantly improves the sustained virological response rate in combination with the standard of care of PEG-IFN and ribavirin among patients with chronic genotype 1 HCV infection (43
). Although the antiviral efficacy of protease inhibitor triple therapy appears to be positive, this regimen still has several challenges, such as anemia, a well-recognized side effect of IFN-based regimens, and the emergence of DAA-related resistant variants due to the high replication rate of the virus and the low fidelity of the NS5B polymerase (44
). Indeed, targeting host factors required for the viral life cycle has been considered a favorable strategy to overcome the genetic variability of the viral genome because the mutation rate of host genes is lower than that of viral genes (45
). Accordingly, combinations of DAAs and host-targeted antivirals may provide alternative regimens to overcome these limitations. In addition to targeting host HO-1 expression, lucidone exhibits synergistic activity against HCV replication in combination with other promising inhibitors against distinct targets of HCV (). In regard to lucidone as a potentially clinically useful drug or adjuvant, assessment of anti-HCV activity assay in vivo
animal mode is required. Recently, Kumar et al. have performed in vivo
experiments to demonstrate anti-inflammatory activity of lucidone against lipopolysaccharide (LPS)-induced inflammation in mice (18
). After the intraperitoneal injection with 200 mg of lucidone/kg, the concentration of lucidone in plasma was 368.5 μg/ml (1,439.4 μM), which is ~29-fold higher than the effective concentration of lucidone (50 μM) on reducing HCV RNA levels by 80% an in vitro
cell-based anti-HCV activity assay. For the development of new drugs, the proper translation from an mouse animal drug dosage to a human dosage is essential. The body surface area normalization method is more commonly used to safely predicate a suitable starting dose for a human clinical trial from animal toxicology data, which incorporated several physiologic parameters between different mammalian species, such as oxygen utilization, caloric expenditure, basal metabolic rate, blood volume, circulating plasma proteins, and renal function (46
). For example, the safe starting dose of lucidone used in the adult human is approximated to be 16.22 mg/kg according to the formula for dose translation by the km
factor. In addition, predication of in vivo
human metabolic drug clearance from in vitro
metabolism data and/or in vivo
animal experiments is an important assessment for clinical studies. For predicting the hepatic clearance of drug, several liver models, such as the well-stirred parallel tube and dispersion, have been used to analyze pharmacokinetic parameters using in vitro
hepatic microsome and isolated hepatocytes from animals or humans (47
). These animal experiments will provide practical information on developing lucidone as an adjuvant in current regimens against HCV in the future.
In summary, we found that lucidone possesses anti-HCV activity. An investigation of the mechanism(s) disclosed that the inhibition of HCV replication by lucidone was because of biliverdin production through Nrf2-mediated HO-1 induction, which indicated the feasibility of using selective HO-1 inducers to improve the efficacy of cellular defense pathways against HCV infection and also inhibit viral protease activity.