Induction of Osteopontin by Dengue Virus-3 Infection in THP-1 Cells: Inhibition of the Synthesis by Brefelamide and Its Derivative

Abstract

Osteopontin (OPN) is a multifunctional matricellular protein produced by a broad range of cells including osteoclasts, macrophages, T cells, endothelial cells, and vascular smooth muscle cells. OPN modulates various physiological and pathological events such as inflammation, wound healing, and bone formation and remodeling. Dengue virus (DENV) infection causes an increase in plasma OPN levels, which is correlated with the severity of symptoms and coagulation abnormalities. DENV infection also induces OPN gene expression in human macrophages. This study investigated the inhibitory effects of brefelamide and its methyl ether derivative on DENV-3 by measuring changes in OPN levels in human THP-1 and 293T cell lines infected at different multiplicities of infection and post-infection time points. OPN mRNA expression and viral RNA were detected by reverse transcriptase quantitative real-time PCR, whereas protein level was determined by enzyme-linked immunosorbent assay. We found that viral copy number was higher in 293T than in THP-1 cells. However, THP-1 constitutively expressed higher levels of OPN mRNA and protein, which were enhanced by DENV-3 infection. Brefelamide and its derivative suppressed OPN production in DENV-3 infected THP-1 cells; the effective doses of these compounds had no effect on uninfected cells, indicating low cytotoxicity. These results suggest that brefelamide and its methyl ether derivative have therapeutic effects in preventing inflammation, coagulopathy, and fibrinolysis caused by OPN upregulation induced by DENV-3 infection.

Inhibition of Osteopontin Synthesis in THP-1 Cells Stimulated with Phorbol 12-Myristate 13-Acetate by Brefelamide Derivatives

“…To analyze the metabolism of OPN products in THP-1 cells, we developed 3 new compounds (C, D, and E). The chemical structure of compound B was previously reported[18]; B had the highest IC50 when compared to C-E based on the transcription assay performed (Table1). In ELISA for FL-OPN, similarly C-E shows lower IC50 values.…”

“…The structure of compound B has already been elucidated ( Figure.1) [18], while those of the other new compounds (C, D, and E) will be described elsewhere (unpublished data). Luciferase expression in A549/OPN-luc cells was dose-dependently suppressed by the compounds; compound B showed the highest IC50 when compared to the other compounds which showed lower IC50 values (Table 1).…”

“…Brefelamide is an aromatic amide isolated from methanol extracts of Dictyostelium brefeldianum and D. giganteum slime mold fruiting bodies [29]; its derivative is referred to as compound B, which contains a methyl ether group (−OCH3) ( Figure 1) [18]. Compounds C, D and E were also synthesized (unpublished data).…”

“…Brefelamide has been reported to inhibit OPN expression in A549 cells at a concentration of 50 µM using an OPN promoter reporter assay [17]. We also reported that brefelamide and its methyl ether derivative (compound B; Figure. 1) suppressed OPN production in DENV-infected THP-1 cells and also inhibited DENV replication [18]. Recently, we developed three novel derivatives which exhibited inhibitory activity at lower concentrations and examined their effect on OPN production in PMA-stimulated THP-1 cells.…”

“…Cells were seeded in 96-well tissue culture plates at a density of 2 × 10 4 /well in 50 µl of complete medium followed by the addition of compounds and PMA) to each well.After incubation for 48 h, 20 µl per well of 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)2-(4sulfophenyl)-2H-tetrazolium salt/phenazine methosulfate solution was added to each well, followed by incubation for 4 h. The absorbance at 492 nm was recorded with an ELISA plate reader.. The IC50 value was determined by identifying the X-axis value corresponding to N-half of the difference between the maximum and minimum absorbance values using GraphPad Prism 7 software (GraphPadInc., San Diego, CA, USA) as described [18]. 4.5.…”