Characterization of anti-HCV activities of N-89 and N-251.

<p>(A) Schematic gene organization of authentic HCV-RNA (HCV-O/RLGE). The positions of four adaptive mutations - Q1112R, P1115L, E1202G, and K1609E - are indicated by a black star. (B) N-89 and N-251 inhibited authentic HCV-RNA replication. The cells harboring HCV-O/RLGE RNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Ikeda4" target="_blank">[19]</a> were treated with N-89 (left panel) and N-251 (right panel) for 72 hrs, followed by real-time LightCycler PCR (black circles in the upper panel) and WST-1 assay (open triangles in the upper panel). The relative value (%) calculated at each point, when the level in non-treated cells was assigned as 100%, is presented here. Data are expressed as the means±standard deviation of triplicate assays. Western blot analysis (lower panels) was performed as described in Fig. 1B. (C) N-89 did not inhibit the HCV-JFH-1 replication. RSc (left panel) and D7 (right panel) cells were inoculated with supernatant from RSc cells replicating JR/C5B/BX-2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Takeda1" target="_blank">[29]</a>. The RL assay was performed as described in Fig. 1B. (D) N-89 (left panel) and N-251 (right panel) did not inhibit the replication of HCV-JFH-1 subgenomic replicon. The RL and WST-1 assays were performed as described in Fig. 1B.</p

Synergistic anti-HCV effects of N-89 or N-251 in combination with IFN-α and/or RBV on HCV-RNA replication in ORL8 cells.

<p>Open symbols in the broken lines show the values expected as an additive anti-HCV effect and closed symbols in the solid lines show the values obtained by the ORL8 assay. ORL8 cells were treated with N-89 (upper panel) or N-251 (lower panel) in combination with IFN-α (A), RBV (B), or IFN-α and RBV (C) for 72 hrs and subjected to RL assay.</p

Anti-HCV activities of N-89 or N-251 in various HCV drug assay systems.

*<p>¹CC₅₀ value (µM),</p>*<p>²EC₅₀ value (µM),</p>*<p>³SI value.</p

Anti-HCV activities of N-89 and N-251 detected in the OR6, ORL8, and ORL11 assays.

<p>(A) Structures of N-89 and N-251. (B) Effects of N-89 on genome-length HCV-RNA replication. OR6, ORL8, and ORL11 cells were treated with N-89 for 72 hrs, followed by RL assay (black circles in the upper panel) and WST-1 assay (open triangles in the upper panel). The relative value (%) calculated at each point, when the level in non-treated cells was assigned as 100%, is presented here. Data are expressed as the means±standard deviation of triplicate assays. Western blot analysis of the treated cells for the HCV Core was also performed (lower panel). β-actin was used as a control for the amount of protein loaded per lane. (C) Effects of N-251 on genome-length HCV-RNA replication. The RL assay, WST-1 assay, and Western blot analysis were performed as described in (B).</p

The anti-HCV action of N-89 was faster than that of IFN-α.

<p>The ORL8 cells were treated with N-89 or IFN-α, and then RL assays were performed at 2 to 72 hrs after the treatment. The relative value (%) calculated at each time point, when the luciferase activity of non-treated cells at 24 hrs was assigned as 100%, is shown. Data are expressed as the means±standard deviation of triplicate assays. The data within 12 hrs after the treatment are shown in the lower panel. *<i>P</i><0.05; **<i>P</i><0.01.<b>The</b> Anti<b>-HCV</b> Activities <b>of N-89 and N-251 were</b> Completely Canceled <b>by VE</b>We previously reported that the antioxidant VE canceled the anti-HCV activities of CsA and three nutrients (β-carotene, vitamin D₂, and linoleic acid) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Yano1" target="_blank">[37]</a>, and demonstrated that the oxidative stress induced by these anti-HCV reagents caused anti-HCV status via activation of the extracellular signal-regulated kinase signaling pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Yano2" target="_blank">[38]</a>. To evaluate this possibility, we examined the effect of VE on N-89 at the EC₉₀ level in the ORL8 assay. CsA and IFN-α were also used as a positive and a negative control, respectively, on the effect of VE in the ORL8 assay. The results revealed that the anti-HCV activities of N-89 and CsA were largely canceled by VE, whereas the activity of IFN-α was not canceled (Fig. 5A). We normalized these results by dividing the RL value obtained in the presence of VE by that in the absence of VE as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Mori2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072519#pone.0072519-Yano1" target="_blank">[37]</a>. The values of N-89 and CsA were 16 and 34, respectively, whereas the value (3.2) of IFN-α was almost the same as that (3.0) of the control (Fig. 5B). Similar results were obtained by using N-251 (Fig. 5C and D). The values of N-251, CsA, and IFN-α were 13, 19, and 4.3, respectively, in comparison with the value (2.3) of the control (Fig. 5D). These results suggest that the induction of oxidative stress is associated with the anti-HCV activity of N-89 or N-251. However, an antimalarial drug, artemisinin, was hardly influenced by co-treatment with VE (Fig. 5E). The value (1.9) of artemisinin was almost the same as that (3.5 or 2.5) of IFN-α or the control, respectively (Fig. 5F). These results were also confirmed by Western blot analysis of HCV Core (Fig. 5G). Therefore, our results suggest that the anti-HCV mechanism of artemisinin is not associated with the induction of oxidative stress, and is distinct from that of N-89 or N-251.</p

The anti-HCV activity of N-89 or N-251 was canceled by addition of VE.

<p>Effect of VE on the anti-HCV activity of N-89 (A), N-251 (C), Artemisinin (E), CsA, or IFN-α at the expected EC₉₀. ORL8 cells were treated with control medium (−), N-89, CsA, or IFN-α in either the absence or presence of VE for 72 hrs. After treatment, an RL assay of harvested ORL8 cell samples was performed. (B, D, and F) The ratio of RL activity in the presence of VE to the RL activity in the absence of VE. The above ratio was calculated from the data of (A, C, and E). The horizontal line indicates the promoting effect of VE alone on HCV-RNA replication as a baseline. (G) Western blot analysis was performed as described in Fig. 1B.</p

OR6 and ORL8 cells were cured by treatment with only N-89.

<p>The treated cells were divided into two plates with or without G418, and then cultured for 2 weeks. The left panels show the cells stained with Coomassie brilliant blue. The right panels show the results of Western blot analysis of the treated and non-treated cells for HCV proteins. Western blot analysis was performed as described in Fig. 1B.</p

EGFP-tagged AdipoR1 protein expression and signaling function in MIN6 cells.

<p>(A) Representative fluorescence images of MIN6 cells transfected with empty vector (control), AdipoR1 or EGFP-tagged AdipoR1 (AdipoR1-EGFP) vectors. The scale bars represent the indicated dimensions. (B) Western blot analysis of cytosolic and membrane fractions of MIN6 cells transfected with control, AdipoR1or AdipoR1-EGFP vectors using anti-AdipoR1 antibody. Expected protein sizes are AdipoR1 (43 kDa) and AdipoR1-EGFP (70 kDa). (C) Akt phosphorylation in MIN6 cells transfected with control, AdipoR1 or AdipoR1-EGFP vectors. MIN6 cells were stimulated with 5μg/ml fAd in serum-free medium after transfections. Representative Western blots for Akt (60 kDa), phospho-Akt (Ser⁴⁷³) (60 kDa) and vinculin (124 kDa), as a loading control, are shown (left panel). Quantified values represent the ratios (compared with control) of phospho-Akt/Akt (right panel). Data are represented as mean ± SEM of four independent experiments. *p < 0.05, by ANOVA followed by Tukey’s honestly significant difference test compared with control.</p

Growth curves and blood glucose of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Growth curves and blood glucose levels of AdipoR1/Akita (n = 14), Akita (n = 11), AdipoR1 (n = 12), and WT (n = 10) mice. (A) Time course of body weight changes of mice between 4 and 10 weeks of age. Blood glucose after (B) an Ad lib feed and (C) a 16 h fast in mice between 4 and 10 weeks of age. Values are expressed as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita mice, nor between AdipoR1 and WT mice, whereas there were significant differences between AdipoR1/Akita vs AdipoR1 (* p<0.05), and Akita and WT (# p<0.05) mice by ANOVA followed by Tukey’s honestly significant difference test.</p

Pancreatic insulin contents and morphological characteristics of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Pancreatic tissue samples were obtained from AdipoR1/Akita, AdipoR1, Akita and WT mice at 15–20 weeks of age. (A) Pancreatic insulin contents of AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice. (B) Insulin positive area per islet area in AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice (upper panel). Quantification was performed on more than 20 islets from each mouse. Representative images of tissues/islet cells immunostained with anti-insulin antibodies (lower panel). Scale bars indicate 50 μm. (C) Representative images of electron micrographs of islet cells in AdipoR1/Akita, AdipoR1, Akita and WT mice. Similar results were obtained from three independent experiments. Scale bars indicate 2 μm. The small boxed areas have been enlarged and are shown in the corresponding large boxes. Arrows indicate insulin secretory granules. Data are shown as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita, or between AdipoR1 and WT, whereas there was significant differences between AdipoR1/Akita and AdipoR1 (* p<0.05), and between Akita and WT (# p<0.05) by ANOVA followed by Tukey’s honestly significant difference test. NS, Not significant.</p