Contrasting Effect of Curcumin on Hepatitis B Virus Replication According to the Hepatoma Cell Line

Abstract

In recent decades, considerable advances have been achieved in the treatment of chronic hepatitis B. However, the currently available drugs have shortcomings. In this context, several natural compounds have been proposed as potential agents to improve either the outcome of antiviral treatment or the progression of chronic infection, with curcumin being one of the most evaluated compounds due to its pleiotropic antiviral activity. The aim of this study was to characterize the effect and mechanism of curcumin on hepatitis B virus (HBV) replication in two different experimental models. Treatment of HepG22.15 and HBV-transfected Huh7 cells with curcumin revealed that the phytochemical differentially modulated HBV replication in both cell lines. In HepG22.15 cells, the addition of curcumin had no effect on viral DNA, pregenomic RNA (pgRNA), and e antigen (HBeAg) levels, while it decreased Precore RNA and s antigen (HBsAg) levels. Conversely, in Huh-7 cells, curcumin significantly increased viral progeny more than tenfold, as well as HBV RNAs and viral antigens. Furthermore, the analysis of the cellular mechanisms associated with the modulation of viral replication revealed that in Huh-7 cells, curcumin-induced cell cycle arrest in the G2/M phase and the modulation of genes involved in proliferation, cell cycle progression, and apoptosis, whereas no changes in cell cycle progression and gene expression were observed in HepG22.15 cells. In conclusion, curcumin elicits a differential cellular response in two hepatoma cell lines, which, in the case of Huh-7 cells, would provide an optimal cellular setting that enhances HBV replication. Therefore, the antiviral effect of this phytochemical remains controversial.

Keywords: HepG22.15; Huh-7; curcumin; hepatitis B virus; replication.

Figure 1

Effects of curcumin in cell viability in HepG22.15 and Huh-7 cells. HepG22.15, Huh-7 non-transfected (−HBV), and HBV transfected Huh-7 (+HBV) cells were incubated for 24 (A), 48 (B), and 72 h (C) in the presence of 0, 10, 20, and 30 μM of curcumin. Cell viability was measured by the MTS assay. Shown values represent the mean ± standard deviation of three independent experiments. * p < 0.05; ** p < 0.005 and *** p < 0.0001.

Figure 2

Effects of curcumin on HBV replicative capacity in HepG22.15 and Huh-7 cells. HepG22.15 and HBV-transfected Huh-7 cells were incubated for 72 h in the presence of 0, 10, and 20 μM of curcumin. (A) Extracellular capsid-associated HBV DNA was quantified by qPCR. Total RNA was extracted, and pgRNA (B) and Precore mRNA (C) levels were analyzed by transcript-specific RT-qPCR. Culture supernatants were harvested, and extracellular levels of HBsAg (D) and HBeAg (E) were determined by ECLIA. Shown values represent the mean ± standard deviation of three independent experiments. * p < 0.05; ** p < 0.005 and *** p < 0.0001.

Figure 3

Effects of curcumin on cell cycle progression in HepG22.15 and Huh-7 cells. HepG22.15 and HBV-transfected Huh-7 cells were incubated for 72 h in the presence of 0 and 20 μM of curcumin, and cell cycle progression was analyzed by flow cytometry (A). The percentage of cells in G0/G1 (B), S (C), and G2/M (D) phases are shown. Shown values represent the mean ± standard deviation of three independent experiments. * p < 0.05; ** p < 0.005 and *** p < 0.0001.

Figure 4

Effects of curcumin in the expression cell cycle, proliferation, and apoptosis-related genes in HepG22.15 and Huh-7 cells. HepG22.15 and HBV-transfected Huh-7 cells were incubated for 72 h in the presence of 0 and 20 μM of curcumin. Total RNA was extracted, and mRNA levels of cell cycle and proliferation-related genes were analyzed by RT-qPCR. Relative expression was calculated using the method of 2^−ΔΔCt. Bars represent the expression levels of cells treated with 20 μM curcumin relativized to control cells (0 μM, dash line). Values shown represent the mean ± standard deviation of three independent experiments. * p < 0.05; ** p < 0.005; *** p < 0.0001.